def flashing_rail_lights(protocol: protocol_api.ProtocolContext,
seconds_per_flash_cycle=1.0):
"""Flash the rail lights on and off in the background.
Source: https://github.com/Opentrons/opentrons/issues/7742
Example usage:
# While the robot is doing nothing for 2 minutes, flash lights quickly.
with flashing_rail_lights(protocol, seconds_per_flash_cycle=0.25):
protocol.delay(minutes=2)
When the ``with`` block exits, the rail lights are restored to their
original state.
Exclusive control of the rail lights is assumed. For example, within the
``with`` block, you must not call `ProtocolContext.set_rail_lights`
yourself, inspect `ProtocolContext.rail_lights_on`, or nest additional
calls to `flashing_rail_lights`.
"""
original_light_status = protocol.rail_lights_on
stop_flashing_event = threading.Event()
def background_loop():
while True:
protocol.set_rail_lights(not protocol.rail_lights_on)
# Wait until it's time to toggle the lights for the next flash or
# we're told to stop flashing entirely, whichever comes first.
got_stop_flashing_event = stop_flashing_event.wait(
timeout=seconds_per_flash_cycle / 2)
if got_stop_flashing_event:
break
background_thread = threading.Thread(
target=background_loop,
name="Background thread for flashing rail \
lights")
try:
if not protocol.is_simulating():
background_thread.start()
yield
finally:
# The ``with`` block might be exiting normally, or it might be exiting
# because something inside it raised an exception.
#
# This accounts for user-issued cancelations because currently
# (2021-05-04), the Python Protocol API happens to implement user-
# issued cancellations by raising an exception from internal API code.
if not protocol.is_simulating():
stop_flashing_event.set()
background_thread.join()
# This is questionable: it may issue a command to the API while the API
# is in an inconsistent state after raising an exception.
protocol.set_rail_lights(original_light_status)
def run(ctx: protocol_api.ProtocolContext):
global robot
robot = ctx
# confirm door is close
if not ctx.is_simulating():
confirm_door_is_closed()
# define tips
tips1000 = [
ctx.load_labware('opentrons_96_filtertiprack_1000ul', slot)
for slot in ['3', '6']
]
tips300 = [ctx.load_labware('opentrons_96_filtertiprack_200ul', '9')]
# define pipettes
p1000 = ctx.load_instrument('p1000_single_gen2',
'left',
tip_racks=tips1000)
p300 = ctx.load_instrument('p300_single_gen2', 'right', tip_racks=tips300)
# check buffer labware type
if BUFFER_LABWARE not in BUFFER_LW_DICT:
raise Exception('Invalid BF_LABWARE. Must be one of the \
following:\nopentrons plastic 50ml tubes')
# load mastermix labware
buffer_rack = ctx.load_labware(BUFFER_LW_DICT[BUFFER_LABWARE], '10',
BUFFER_LABWARE)
# check mastermix tube labware type
if DESTINATION_LABWARE not in DESTINATION_LW_DICT:
raise Exception('Invalid DESTINATION_LABWARE. Must be one of the \
following:\nopentrons plastic 2ml tubes')
# load elution labware
dest_racks = [
ctx.load_labware(DESTINATION_LW_DICT[DESTINATION_LABWARE], slot,
'Destination tubes labware ' + str(i + 1))
for i, slot in enumerate(['4', '1', '5', '2'])
]
# setup sample sources and destinations
bf_tubes = buffer_rack.wells()[:4]
number_racks = math.ceil(NUM_SAMPLES / len(dest_racks[0].wells()))
# dest_sets is a list of lists. Each list is the destination well for each rack
# example: [[tube1,tube2,...tube24](first rack),[tube1,tube2(second rack),...]
dest_sets = [[tube for rack in dest_racks for tube in rack.wells()
][:NUM_SAMPLES][i * len(dest_racks[0].wells()):(i + 1) *
len(dest_racks[0].wells())]
for i in range(number_racks)]
# transfer buffer to tubes
for bf_tube, dests in zip(bf_tubes, dest_sets):
transfer_buffer(bf_tube, dests, VOLUME_BUFFER, p1000, tips1000)
# track final used tip
save_tip_info(p1000)
finish_run()
def run(ctx: protocol_api.ProtocolContext):
global robot
robot = ctx
# confirm door is closed
if not ctx.is_simulating():
confirm_door_is_closed(ctx)
tips1000 = [
ctx.load_labware('opentrons_96_filtertiprack_1000ul', 3,
'1000µl tiprack')
]
# load pipette
p1000 = ctx.load_instrument('p1000_single_gen2',
'left',
tip_racks=tips1000)
# check source (LYSATE) labware type
if LYSATE_LABWARE not in LY_LW_DICT:
raise Exception('Invalid LYSATE_LABWARE. Must be one of the \
following:\nopentrons plastic 2ml tubes')
# load LYSATE labware
if 'plate' in LYSATE_LABWARE:
source_racks = ctx.load_labware(LY_LW_DICT[LYSATE_LABWARE], '1',
'RNA LYSATE labware')
else:
source_racks = [
ctx.load_labware(LY_LW_DICT[LYSATE_LABWARE], slot,
'sample LYSATE labware ' + str(i + 1))
for i, slot in enumerate(['4', '1', '5', '2'])
]
# check plate
if PLATE_LABWARE not in PL_LW_DICT:
raise Exception('Invalid PLATE_LABWARE. Must be one of the \
following:\nopentrons deep generic well plate\nnest deep generic well plate\nvwr deep generic well plate'
)
# load pcr plate
wells_plate = ctx.load_labware(PL_LW_DICT[PLATE_LABWARE], 10,
'sample LYSATE well plate ')
# setup samples
#sources, dests = get_source_dest_coordinates(LYSATE_LABWARE, source_racks, wells_plate)
sources = [tube for s in source_racks for tube in s.wells()][:NUM_SAMPLES]
dests = wells_plate.wells()[:NUM_SAMPLES]
# transfer
transfer_samples(LYSATE_LABWARE, VOLUME_LYSATE, sources, dests, p1000,
tips1000)
# track final used tip
save_tip_info()
finish_run()
def run(ctx: protocol_api.ProtocolContext):
# Turn on rail lights and pause program so user can load robot deck.
# ctx.set_rail_lights(True)
# ctx.pause("Load Labware onto robot deck and click resume when ready to continue")
# ctx.home()
ctx.set_rail_lights(False)
# TSV file location on OT-2
tsv_file_path = "{0}var{0}lib{0}jupyter{0}notebooks{0}ProcedureFile.tsv".format(
os.sep)
if not os.path.isfile(tsv_file_path):
# Temp TSV file location on Win10 Computers for simulation
tsv_file_path = "C:{0}Users{0}{1}{0}Documents{0}TempTSV.tsv".format(
os.sep, os.getlogin())
sample_parameters, args = Utilities.parse_sample_template(tsv_file_path)
labware_dict, left_tiprack_list, right_tiprack_list = Utilities.labware_parsing(
args, ctx)
# Pipettes
left_pipette = ctx.load_instrument(args.LeftPipette,
'left',
tip_racks=left_tiprack_list)
right_pipette = ctx.load_instrument(args.RightPipette,
'right',
tip_racks=right_tiprack_list)
# Set the location of the first tip in box.
left_pipette.starting_tip = left_tiprack_list[0].wells_by_name()[
args.LeftPipetteFirstTip]
right_pipette.starting_tip = right_tiprack_list[0].wells_by_name()[
args.RightPipetteFirstTip]
# Make sample dilutions. Calculate sample and water volumes.
sample_data_dict, aspirated_water_vol = \
process_samples(args, ctx, sample_parameters, labware_dict, left_pipette, right_pipette)
# Dispense Water
aspirated_water_vol = \
dispense_water(args, sample_data_dict, labware_dict, left_pipette, right_pipette, aspirated_water_vol)
# Dispense Samples
aspirated_water_vol = \
dispense_samples(args, sample_data_dict, left_pipette, right_pipette, aspirated_water_vol)
# Dispense PCR Reagents.
dispense_pcr_reagents(args, labware_dict, left_pipette, right_pipette,
aspirated_water_vol, sample_data_dict)
if not ctx.is_simulating():
os.remove(tsv_file_path)
def run(ctx: protocol_api.ProtocolContext):
ctx.comment("Begin {}".format(metadata['protocolName']))
# Turn on rail lights and pause program so user can load robot deck.
# ctx.set_rail_lights(True)
# ctx.pause("Load Labware onto robot deck and click resume when ready to continue")
# ctx.home()
ctx.set_rail_lights(False)
# TSV file location on OT-2
tsv_file_path = "{0}var{0}lib{0}jupyter{0}notebooks{0}ProcedureFile.tsv".format(
os.sep)
if not os.path.isfile(tsv_file_path):
# Temp TSV file location on Win10 Computers for simulation
tsv_file_path = "C:{0}Users{0}{1}{0}Documents{0}TempTSV.tsv".format(
os.sep, os.getlogin())
sample_parameters, args = Utilities.parse_sample_template(tsv_file_path)
labware_dict, left_tiprack_list, right_tiprack_list = Utilities.labware_parsing(
args, ctx)
# Pipettes
left_pipette = ctx.load_instrument(args.LeftPipette,
'left',
tip_racks=left_tiprack_list)
right_pipette = ctx.load_instrument(args.RightPipette,
'right',
tip_racks=right_tiprack_list)
# Set the location of the first tip in box.
with suppress(IndexError):
left_pipette.starting_tip = left_tiprack_list[0].wells_by_name()[
args.LeftPipetteFirstTip]
with suppress(IndexError):
right_pipette.starting_tip = right_tiprack_list[0].wells_by_name()[
args.RightPipetteFirstTip]
# Dispense Samples and primers
sample_dest_dict = dispense_samples(args, sample_parameters, labware_dict,
left_pipette, right_pipette)
# Add PCR mix to each destination well.
add_pcr_mix(args, labware_dict, sample_dest_dict, left_pipette,
right_pipette)
if not ctx.is_simulating():
os.remove(tsv_file_path)
ctx.comment("Program End")
def run(ctx: protocol_api.ProtocolContext):
#Please replace what is between the quotation marks!!
NEW_SERIAL_NUMBER = "OT2CEP20201214B12"
ctx.comment(f"Setting serial number to {NEW_SERIAL_NUMBER}.")
if not ctx.is_simulating():
with open("/var/serial", "w") as serial_number_file:
serial_number_file.write(NEW_SERIAL_NUMBER + "\n")
with open("/etc/machine-info", "w") as serial_number_file:
serial_number_file.write(f"DEPLOYMENT=production\nPRETTY_HOSTNAME={NEW_SERIAL_NUMBER}\n")
with open("/etc/hostname", "w") as serial_number_file:
serial_number_file.write(NEW_SERIAL_NUMBER + "\n")
os.sync()
ctx.comment("Done.")
def run(ctx: protocol_api.ProtocolContext):
#Please replace what is between the quotation marks!!
NEW_SERIAL_NUMBER = "set_your_serial_number_here"
ctx.comment(f"Setting serial number to {NEW_SERIAL_NUMBER}.")
if not ctx.is_simulating():
with open("/var/serial", "w") as serial_number_file:
serial_number_file.write(NEW_SERIAL_NUMBER + "\n")
with open("/etc/machine-info", "w") as serial_number_file:
serial_number_file.write(f"DEPLOYMENT=production\nPRETTY_HOSTNAME={NEW_SERIAL_NUMBER}\n")
with open("/etc/hostname", "w") as serial_number_file:
serial_number_file.write(NEW_SERIAL_NUMBER + "\n")
os.sync()
ctx.comment("Done.")# This script sets an OT-2's serial number. It's meant to be used after you
def run(ctx: protocol_api.ProtocolContext):
# load labware
s_racks = [
ctx.load_labware('opentrons_15_tuberack_falcon_15ml_conical', '4')
]
d_plate = ctx.load_labware('nest_96_wellplate_200ul_flat', '1',
'96-wellplate sample plate')
tips300 = [ctx.load_labware('opentrons_96_filtertiprack_200ul', '5')]
# load pipette
p300 = ctx.load_instrument('p300_single_gen2', 'left', tip_racks=tips300)
# transfer sample
p300.transfer(SAMPLE_VOLUME,
s_racks.wells()['A1'],
d_plate.columns()['5'],
air_gap=20,
mix_before=(2, 200),
new_tip='never')
p300.air_gap(20)
p300.drop_tip()
ctx.comment('Terminado.')
# track final used tip
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
data = {'tips300': tip_log['count'][p300]}
with open(tip_file_path, 'w') as outfile:
json.dump(data, outfile)
def run(ctx: protocol_api.ProtocolContext):
STEP = 0
STEPS = { # Dictionary with STEP activation, description and times
1: {'Execute': True, 'description': 'Mix and move samples ('+str(VOLUME_SAMPLE)+'ul)'}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
if not ctx.is_simulating():
folder_path = '/var/lib/jupyter/notebooks/'+run_id
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/StationA_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse, delay):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.delay = delay
# Reagents and their characteristics
Samples = Reagent(name = 'Samples',
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
rinse = False,
delay = 0
)
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('CONTROL SPACES: ' + str(NUM_CONTROL_SPACES))
ctx.comment('NUM SAMPLES: ' + str(NUM_REAL_SAMPLES))
ctx.comment('###############################################')
ctx.comment(' ')
##################
# Custom functions
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = 2, blow_out = True, mix_height = 0,
x_offset = x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z = -2))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -10)
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
x_offset, source_height = 5):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height <= 0:
mix_height = 3
pipet.aspirate(1, location = location.bottom(
z = source_height).move(Point(x = x_offset[0])), rate = reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol, location = location.bottom(
z = source_height).move(Point(x = x_offset[0])), rate = reagent.flow_rate_aspirate)
pipet.dispense(vol, location = location.bottom(
z = mix_height).move(Point(x = x_offset[1])), rate = reagent.flow_rate_dispense)
pipet.dispense(1, location = location.bottom(
z = mix_height).move(Point(x = x_offset[1])), rate = reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z = -2)) # Blow out
def generate_source_table(source):
'''
Concatenate the wells frome the different origin racks
'''
num_cols = math.ceil(num_samples / 8)
s = []
for i in range(num_cols):
if i < 6:
s += source[0].columns()[i] + source[1].columns()[i]
else:
s += source[2].columns()[i - 6] + source[3].columns()[i - 6]
return s
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
####################################
# load labware and modules
####################################
# Load Sample racks
if num_samples <= 48:
rack_num = 2
ctx.comment('Used source racks are ' + str(rack_num))
else:
rack_num = 4
source_racks = [ctx.load_labware(
'opentrons_24_tuberack_nest_2ml_snapcap', slot,
'source tuberack with snapcap' + str(i + 1)) for i, slot in enumerate(['5', '2', '6', '3'][:rack_num])
]
##################################
# Destination plate
dest_plate = ctx.load_labware(
'nest_96_wellplate_2ml_deep', '1',
'NEST 96 Deepwell Plate 2mL')
####################################
# Load tip_racks
tips1000 = [ctx.load_labware(
'opentrons_96_filtertiprack_1000ul', slot,
'1000µl filter tiprack') for slot in ['7']]
################################################################################
# setup samples and destinations
sample_sources_full = generate_source_table(source_racks)
sample_sources = sample_sources_full[NUM_CONTROL_SPACES:num_samples]
destinations = dest_plate.wells()[NUM_CONTROL_SPACES:num_samples]
p1000 = ctx.load_instrument(
'p1000_single_gen2', 'right',
tip_racks = tips1000) # load P1000 pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {p1000: 0},
'maxes': {p1000: len(tips1000) * 96}
}
############################################################################
# STEP 1: MIX AND MOVE SAMPLES
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
start = datetime.now()
for s, d in zip(sample_sources, destinations):
if not p1000.hw_pipette['has_tip']:
pick_up(p1000)
# Mix the sample BEFORE dispensing
if NUM_MIXES > 0:
custom_mix(p1000, reagent = Samples, location = s, vol = volume_mix,
rounds = NUM_MIXES, blow_out = True, mix_height = 15, x_offset = x_offset)
move_vol_multichannel(p1000, reagent = Samples, source = s, dest = d,
vol = VOLUME_SAMPLE, air_gap_vol = air_gap_vol_sample, x_offset = x_offset,
pickup_height = 3, rinse = Samples.rinse, disp_height = -10,
blow_out = True, touch_tip = True)
p1000.drop_tip(home_after = False)
tip_track['counts'][p1000] += 1
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
# from opentrons.drivers.rpi_drivers import gpio
#if not ctx.is_simulating():
#os.system('mpg123 -f -14000 /var/lib/jupyter/notebooks/final.mp3')
if not ctx.is_simulating():
fname = ' /var/lib/jupyter/notebooks/finished_process_esp.mp3'
if os.path.isfile(fname) is True:
subprocess.run(
['mpg123', fname],
stdout=subprocess.PIPE,
stderr=subprocess.PIPE
)
else:
ctx.comment(f"Sound file does not exist. Call the technician")
for i in range(3):
ctx._hw_manager.hardware.set_lights(rails = False)
ctx._hw_manager.hardware.set_lights(button = (1, 0 ,0))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails = True)
ctx._hw_manager.hardware.set_lights(button = (0, 0 ,1))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button = (0, 1 ,0))
ctx.comment('Finished! \nMove deepwell plate (slot 1) to Station B for extraction protocol')
ctx.comment('Used p1000 tips in total: ' + str(tip_track['counts'][p1000]))
ctx.comment('Used p1000 racks in total: ' + str(tip_track['counts'][p1000] / 96))
def run(ctx: protocol_api.ProtocolContext):
from opentrons.drivers.rpi_drivers import gpio
gpio.set_rail_lights(False) #Turn off lights (termosensible reagents)
ctx.comment('Actual used columns: ' + str(num_cols))
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {'Execute': True, 'description': 'Transfer MMIX'},
2: {'Execute': True, 'description': 'Transfer elution'}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
folder_path = '/var/lib/jupyter/notebooks/'+run_id'
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/KC_qPCR_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
reagent_reservoir_volume, delay, num_wells, h_cono, v_fondo,
tip_recycling = 'none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused=[]
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
MMIX = Reagent(name = 'Master Mix',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = volume_mmix_available,
num_wells = 2, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
Samples = Reagent(name='Samples',
rinse=False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume=50,
delay=0,
num_wells=num_cols, # num_cols comes from available columns
h_cono=0,
v_fondo=0
)
MMIX.vol_well = MMIX.vol_well_original
Samples.vol_well = Samples.vol_well_original
##################
# Custom functions
def divide_destinations(l, n):
# Divide the list of destinations in size n lists.
for i in range(0, len(l), n):
yield l[i:i + n]
def distribute_custom(pipette, volume, src, dest, waste_pool, pickup_height, extra_dispensal, disp_height=0):
# Custom distribute function that allows for blow_out in different location and adjustement of touch_tip
pipette.aspirate((len(dest) * volume) +
extra_dispensal, src.bottom(pickup_height))
pipette.touch_tip(speed=20, v_offset=-5)
pipette.move_to(src.top(z=5))
pipette.aspirate(20) # air gap
for d in dest:
pipette.dispense(20, d.top())
drop = d.top(z = disp_height)
pipette.dispense(volume, drop)
pipette.move_to(d.top(z=5))
pipette.aspirate(20) # air gap
try:
pipette.blow_out(waste_pool.wells()[0].bottom(pickup_height + 3))
except:
pipette.blow_out(waste_pool.bottom(pickup_height + 3))
return (len(dest) * volume)
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = 2, blow_out = True, mix_height = 0,
x_offset = x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s, rate = reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z = -2))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -5, radius = 0.9)
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
x_offset, source_height = 3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
pipet.dispense(vol, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
pipet.dispense(1, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
def calc_height(reagent, cross_section_area, aspirate_volume, min_height = 0.5, extra_volume = 50):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume + extra_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
####################################
# load labware and modules
# 24 well rack
tuberack = ctx.load_labware(
'opentrons_24_aluminumblock_generic_2ml_screwcap', '2',
'Bloque Aluminio opentrons 24 screwcaps 2000 µL ')
############################################
# tempdeck
tempdeck = ctx.load_module('tempdeck', '4')
tempdeck.set_temperature(temperature)
##################################
# qPCR plate - final plate, goes to PCR
qpcr_plate = tempdeck.load_labware(
'abi_fast_qpcr_96_alum_opentrons_100ul',
'chilled qPCR final plate')
##################################
# Sample plate - comes from B
source_plate = ctx.load_labware(
"kingfisher_std_96_wellplate_550ul", '1',
'chilled KF plate with elutions (alum opentrons)')
samples = source_plate.wells()[:NUM_SAMPLES]
##################################
# Load Tipracks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['5']
]
tips200 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['6']
]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
MMIX.reagent_reservoir = tuberack.rows()[0][:MMIX.num_wells] # 1 row, 2 columns (first ones)
ctx.comment('Wells in: '+ str(tuberack.rows()[0][:MMIX.num_wells]) + ' element: '+str(MMIX.reagent_reservoir[MMIX.col]))
# setup up sample sources and destinations
samples = source_plate.wells()[:NUM_SAMPLES]
samples_multi = source_plate.rows()[0][:num_cols]
pcr_wells = qpcr_plate.wells()[:NUM_SAMPLES]
pcr_wells_multi = qpcr_plate.rows()[0][:num_cols]
# Divide destination wells in small groups for P300 pipette
dests = list(divide_destinations(pcr_wells, size_transfer))
# pipettes
m20 = ctx.load_instrument(
'p20_multi_gen2', mount='right', tip_racks=tips20)
p300 = ctx.load_instrument(
'p300_single_gen2', mount='left', tip_racks=tips200)
# used tip counter and set maximum tips available
tip_track = {
'counts': {p300: 0,
m20: 0}
}
############################################################################
# STEP 1: Transfer Master MIX
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
p300.pick_up_tip()
used_vol=[]
for dest in dests:
aspirate_volume=volume_mmix * len(dest) + extra_dispensal
[pickup_height,col_change]=calc_height(MMIX, area_section_screwcap, aspirate_volume)
used_vol_temp = distribute_custom(
p300, volume = volume_mmix, src = MMIX.reagent_reservoir[MMIX.col], dest = dest,
waste_pool = MMIX.reagent_reservoir[MMIX.col], pickup_height = pickup_height,
extra_dispensal = extra_dispensal)
used_vol.append(used_vol_temp)
p300.drop_tip()
tip_track['counts'][p300]+=1
#MMIX.unused_two = MMIX.vol_well
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: TRANSFER Samples
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('pcr_wells')
#Loop over defined wells
for s, d in zip(samples_multi, pcr_wells_multi):
m20.pick_up_tip()
#Source samples
move_vol_multichannel(m20, reagent = Samples, source = s, dest = d,
vol = volume_sample, air_gap_vol = air_gap_sample, x_offset = x_offset,
pickup_height = 0.2, disp_height = -10, rinse = False,
blow_out=True, touch_tip=False)
m20.drop_tip()
tip_track['counts'][m20]+=8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
gpio.set_rail_lights(False)
time.sleep(2)
#os.system('mpg123 -f -8000 /var/lib/jupyter/notebooks/toreador.mp3 &')
for i in range(3):
gpio.set_rail_lights(False)
gpio.set_button_light(1, 0, 0)
time.sleep(0.3)
gpio.set_rail_lights(True)
gpio.set_button_light(0, 0, 1)
time.sleep(0.3)
gpio.set_rail_lights(False)
gpio.set_button_light(0, 1, 0)
ctx.comment('Finished! \nMove plate to PCR')
if STEPS[1]['Execute'] == True:
total_used_vol = np.sum(used_vol)
total_needed_volume = total_used_vol
ctx.comment('Total Master Mix used volume is: ' + str(total_used_vol) + '\u03BCl.')
ctx.comment('Needed Master Mix volume is ' +
str(total_needed_volume + extra_dispensal*len(dests)) +'\u03BCl')
ctx.comment('Used Master Mix volumes per run are: ' + str(used_vol) + '\u03BCl.')
ctx.comment('Master Mix Volume remaining in tubes is: ' +
format(np.sum(MMIX.unused)+extra_dispensal*len(dests)+MMIX.vol_well) + '\u03BCl.')
ctx.comment('200 ul Used tips in total: ' + str(tip_track['counts'][p300]))
ctx.comment('200 ul Used racks in total: ' + str(tip_track['counts'][p300] / 96))
if STEPS[2]['Execute'] == True:
ctx.comment('20 ul Used tips in total: ' + str(tip_track['counts'][m20]))
ctx.comment('20 ul Used racks in total: ' + str(tip_track['counts'][m20] / 96))
def run(ctx: protocol_api.ProtocolContext):
global robot
robot = ctx
# confirm door is close
if not ctx.is_simulating():
confirm_door_is_closed()
# load labware and modules
## ELUTION LABWARE
if ELUTION_LABWARE not in ELUTION_LW_DICT:
raise Exception('Invalid ELUTION_LABWARE. Must be one of the \
following:\nopentrons aluminum biorad plate\nopentrons aluminum nest plate')
elution_plate = ctx.load_labware(
ELUTION_LW_DICT[ELUTION_LABWARE], '1',
'elution plate')
## MAGNETIC PLATE LABWARE
magdeck = ctx.load_module('magdeck', '10')
magdeck.disengage()
if MAGPLATE_LABWARE not in MAGPLATE_LW_DICT:
raise Exception('Invalid MAGPLATE_LABWARE. Must be one of the \
following:\nopentrons deep generic well plate\nnest deep generic well plate\nvwr deep generic well plate')
magplate = magdeck.load_labware(MAGPLATE_LW_DICT[MAGPLATE_LABWARE])
## WASTE LABWARE
if WASTE_LABWARE not in WASTE_LW_DICT:
raise Exception('Invalid WASTE_LABWARE. Must be one of the \
following:\nnest 1 reservoir plate')
waste = ctx.load_labware(
WASTE_LW_DICT[WASTE_LABWARE], '11', 'waste reservoir').wells()[0].top(-10)
## REAGENT RESERVOIR
if REAGENT_LABWARE not in REAGENT_LW_DICT:
raise Exception('Invalid REAGENT_LABWARE. Must be one of the \
following:\nnest 12 reservoir plate')
reagent_res = ctx.load_labware(
REAGENT_LW_DICT[REAGENT_LABWARE], '7', 'reagent reservoir')
## TIPS
# using standard tip definition despite actually using filter tips
# so that the tips can accommodate ~220µl per transfer for efficiency
tips300 = [
ctx.load_labware(
'opentrons_96_tiprack_300ul', slot, '200µl filter tiprack')
for slot in ['2', '3', '5', '6', '9','4']
]
tips1000 = [
ctx.load_labware('opentrons_96_filtertiprack_1000ul', slot,
'1000µl filter tiprack')
for slot in ['8']
]
# reagents and samples
num_cols = math.ceil(NUM_SAMPLES/8)
mag_samples_m = magplate.rows()[0][:num_cols]
mag_samples_s = magplate.wells()[:NUM_SAMPLES]
elution_samples_m = elution_plate.rows()[0][:num_cols]
elution_buffer = reagent_res.wells()[0]
bead_buffer = reagent_res.wells()[1:5]
wash_sets = [reagent_res.wells()[i:i+2] for i in [5, 7, 9]]
# pipettes
m300 = ctx.load_instrument('p300_multi_gen2', 'left', tip_racks=tips300)
p1000 = ctx.load_instrument('p1000_single_gen2', 'right',
tip_racks=tips1000)
m300.flow_rate.aspirate = 150
m300.flow_rate.dispense = 300
m300.flow_rate.blow_out = 300
p1000.flow_rate.aspirate = 100
p1000.flow_rate.dispense = 1000
p1000.flow_rate.blow_out = 1000
if(DISPENSE_BEADS):
# premix, transfer, and mix magnetic beads with sample
## bead dests depending on number of samples
bead_dests = bead_buffer[:math.ceil(num_cols/4)]
dispense_beads(bead_dests,mag_samples_m,m300,tips300)
else:
# Mix bead
mix_beads(7, mag_samples_m,m300,tips300)
# incubate off and on magnet
ctx.delay(minutes=5, msg='Incubating off magnet for 5 minutes.')
## First incubate on magnet.
magdeck.engage(height_from_base=22)
ctx.delay(minutes=5, msg='Incubating on magnet for 5 minutes.')
# remove supernatant with P1000
remove_supernatant(mag_samples_s,waste,p1000,tips1000)
# 3x washes
wash(wash_sets,mag_samples_m,waste,magdeck,m300,tips300)
# elute samples
magdeck.disengage()
elute_samples(mag_samples_m,elution_samples_m,elution_buffer,magdeck,m300,tips300)
# track final used tip
save_tip_info()
magdeck.disengage()
finish_run()
def run(ctx: protocol_api.ProtocolContext):
global MM_TYPE
# check source (elution) labware type
source_plate = ctx.load_labware(
'opentrons_96_aluminumblock_nest_wellplate_100ul', '1',
'chilled elution plate on block from Station B')
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['3', '6', '8', '9', '10', '11']
]
tips300 = [ctx.load_labware('opentrons_96_filtertiprack_200ul', '2')]
tempdeck = ctx.load_module('Temperature Module Gen2', '4')
pcr_plate = tempdeck.load_labware(
'opentrons_96_aluminumblock_nest_wellplate_100ul', 'PCR plate')
mm_strips = ctx.load_labware(
'opentrons_96_aluminumblock_nest_wellplate_100ul', '7',
'mastermix strips')
tempdeck.set_temperature(4)
tube_block = ctx.load_labware(
'opentrons_24_aluminumblock_nest_2ml_screwcap', '5',
'2ml screw tube aluminum block for mastermix + controls')
# pipette
m20 = ctx.load_instrument('p20_multi_gen2', 'right', tip_racks=tips20)
p300 = ctx.load_instrument('p300_single_gen2', 'left', tip_racks=tips300)
# setup up sample sources and destinations
num_cols = math.ceil(NUM_SAMPLES/8)
sources = source_plate.rows()[0][:num_cols]
sample_dests = pcr_plate.rows()[0][:num_cols]
tip_log = {'count': {}}
folder_path = '/data/C'
tip_file_path = folder_path + '/tip_log.json'
if TIP_TRACK and not ctx.is_simulating():
if os.path.isfile(tip_file_path):
with open(tip_file_path) as json_file:
data = json.load(json_file)
if 'tips20' in data:
tip_log['count'][m20] = data['tips20']
else:
tip_log['count'][m20] = 0
if 'tips300' in data:
tip_log['count'][p300] = data['tips300']
else:
tip_log['count'][p300] = 0
else:
tip_log['count'] = {m20: 0, p300: 0}
else:
tip_log['count'] = {m20: 0, p300: 0}
tip_log['tips'] = {
m20: [tip for rack in tips20 for tip in rack.rows()[0]],
p300: [tip for rack in tips300 for tip in rack.wells()]
}
tip_log['max'] = {
pip: len(tip_log['tips'][pip])
for pip in [m20, p300]
}
def pick_up(pip):
nonlocal tip_log
if tip_log['count'][pip] == tip_log['max'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_log['count'][pip] = 0
pip.pick_up_tip(tip_log['tips'][pip][tip_log['count'][pip]])
tip_log['count'][pip] += 1
""" mastermix component maps """
mm_tube = tube_block.wells()[0]
mm_dict = {
'volume': 15,
'components': {
tube: vol for tube, vol in zip(tube_block.columns()[1][:3],
[6.25, 1.25, 7.5])
}
}
vol_overage = 1.2 if NUM_SAMPLES > 48 else 1.1 # decrease overage for small sample number
total_mm_vol = mm_dict['volume']*(NUM_SAMPLES+2)*vol_overage
# translate total mastermix volume to starting height
r = mm_tube.diameter/2
mm_height = total_mm_vol/(math.pi*(r**2)) - 5
def h_track(vol):
nonlocal mm_height
dh = 1.1*vol/(math.pi*(r**2)) # compensate for 10% theoretical volume loss
mm_height = mm_height - dh if mm_height - dh > 2 else 2 # stop at 2mm above mm tube bottom
return mm_tube.bottom(mm_height)
if PREPARE_MASTERMIX:
vol_overage = 1.2 if NUM_SAMPLES > 48 else 1.1
for i, (tube, vol) in enumerate(mm_dict['components'].items()):
comp_vol = vol*(NUM_SAMPLES)*vol_overage
pick_up(p300)
num_trans = math.ceil(comp_vol/160)
vol_per_trans = comp_vol/num_trans
for _ in range(num_trans):
p300.air_gap(20)
p300.aspirate(vol_per_trans, tube)
ctx.delay(seconds=3)
p300.touch_tip(tube)
p300.air_gap(20)
p300.dispense(20, mm_tube.top()) # void air gap
p300.dispense(vol_per_trans, mm_tube.bottom(2))
p300.dispense(20, mm_tube.top()) # void pre-loaded air gap
p300.blow_out(mm_tube.top())
p300.touch_tip(mm_tube)
if i < len(mm_dict['components'].items()) - 1: # only keep tip if last component and p300 in use
p300.drop_tip()
mm_total_vol = mm_dict['volume']*(NUM_SAMPLES)*vol_overage
if not p300.hw_pipette['has_tip']: # pickup tip with P300 if necessary for mixing
pick_up(p300)
mix_vol = mm_total_vol / 2 if mm_total_vol / 2 <= 200 else 200 # mix volume is 1/2 MM total, maxing at 200µl
mix_loc = mm_tube.bottom(20) if NUM_SAMPLES > 48 else mm_tube.bottom(5)
p300.mix(7, mix_vol, mix_loc)
p300.blow_out(mm_tube.top())
p300.touch_tip()
# transfer mastermix to strips
mm_strip = mm_strips.columns()[0]
if not p300.hw_pipette['has_tip']:
pick_up(p300)
for i, well in enumerate(mm_strip):
if NUM_SAMPLES % 8 == 0 or i < NUM_SAMPLES % 8:
vol = num_cols*mm_dict['volume']*((vol_overage-1)/2+1)
else:
vol = (num_cols-1)*mm_dict['volume']*((vol_overage-1)/2+1)
p300.transfer(vol, mm_tube, well, new_tip='never')
p300.drop_tip()
# transfer mastermix to plate
mm_vol = mm_dict['volume']
pick_up(m20)
m20.transfer(mm_vol, mm_strip[0].bottom(0.5), sample_dests,
new_tip='never')
m20.drop_tip()
# transfer samples to corresponding locations
for s, d in zip(sources, sample_dests):
pick_up(m20)
m20.transfer(SAMPLE_VOL, s.bottom(2), d.bottom(2), new_tip='never')
m20.mix(1, 10, d.bottom(2))
m20.blow_out(d.top(-2))
m20.aspirate(5, d.top(2)) # suck in any remaining droplets on way to trash
m20.drop_tip()
# track final used tip
if TIP_TRACK and not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
data = {
'tips20': tip_log['count'][m20],
'tips300': tip_log['count'][p300]
}
with open(tip_file_path, 'w') as outfile:
json.dump(data, outfile)
def run(ctx: protocol_api.ProtocolContext):
ctx.comment('Actual used columns: ' + str(num_cols))
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {'Execute': True, 'description': 'Add 300 ul Wash Buffer 1 - Round 1'},
2: {'Execute': True, 'description': 'Add 300 ul Wash Buffer 1 - Round 2'},
3: {'Execute': True, 'description': 'Add 450 ul Wash Buffer 2 - Round 1'},
4: {'Execute': True, 'description': 'Add 450 ul Wash Buffer 2 - Round 2'},
5: {'Execute': True, 'description': 'Add 50 ul Elution Buffer'},
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
folder_path = '/var/lib/jupyter/notebooks/'+run_id
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/KB_PlateFilling_pathogen_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
reagent_reservoir_volume, delay, num_wells, h_cono, v_fondo,
tip_recycling = 'none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay #Delay of reagent in dispense
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused=[]
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
WashBuffer1 = Reagent(name='Wash Buffer 1',
flow_rate_aspirate=0.75,
flow_rate_dispense=1,
rinse=True,
delay=2,
reagent_reservoir_volume=100000,
num_wells=1,
h_cono=0,
v_fondo=0) # Flat surface
WashBuffer2 = Reagent(name='Wash Buffer 1',
flow_rate_aspirate=0.75,
flow_rate_dispense=1,
rinse=True,
delay=2,
reagent_reservoir_volume=100000,
num_wells=1,
h_cono=0,
v_fondo=0) # Flat surface
ElutionBuffer = Reagent(name='Elution Buffer',
flow_rate_aspirate=1,
flow_rate_dispense=1,
rinse=False,
delay=0,
reagent_reservoir_volume=50*NUM_SAMPLES,
num_wells=1,
h_cono=1.95,
v_fondo=695) # Prismatic
WashBuffer1.vol_well = WashBuffer1.vol_well_original
WashBuffer2.vol_well = WashBuffer2.vol_well_original
ElutionBuffer.vol_well = ElutionBuffer.vol_well_original
##################
# Custom functions
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = 2, blow_out = True, mix_height = 0,
x_offset = x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s, rate = reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z = -2))
if touch_tip == True:
pipet.touch_tip(radius=0.9, speed = 20, v_offset = -5) #radius here is 0.9
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
x_offset, source_height = 3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
pipet.dispense(vol, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
pipet.dispense(1, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
def calc_height(reagent, cross_section_area, aspirate_volume, min_height = 0.5, extra_volume = 50):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume + extra_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
##########
def find_side(col):
'''
Detects if the current column has the magnet at its left or right side
'''
if col % 2 == 0:
side = -1 # left
else:
side = 1
return side
####################################
# load labware and modules
# 12 well rack
####################################
reagent_res = ctx.load_labware(
'nest_12_reservoir_15ml', '3', 'Reservoir 12 channel, column 1')
# WashBuffer1 reservoir
####################################
WashBuffer1_reservoir = ctx.load_labware(
'nalgene_1_reservoir_300000ul', '2', 'Ethanol 80% reservoir')
# WashBuffer2 reservoir
####################################
WashBuffer2_reservoir = ctx.load_labware(
'nalgene_1_reservoir_300000ul', '11', 'WashBuffer reservoir')
# Wash Buffer 1 300ul Deepwell plate
############################################
WashBuffer1_300ul_plate1 = ctx.load_labware(
'kf_96_wellplate_2400ul', '1', 'Wash Buffer 1 Deepwell plate 1')
# Wash Buffer 1 300ul Deepwell plate
############################################
WashBuffer1_300ul_plate2 = ctx.load_labware(
'kf_96_wellplate_2400ul', '4', 'Wash Buffer 1 Deepwell plate 2')
# Wash Buffer 2 450ul Deepwell plate
############################################
WashBuffer2_450ul_plate1 = ctx.load_labware(
'kf_96_wellplate_2400ul', '7', 'Wash Buffer 2 Deepwell plate 1')
# Wash Buffer 2 450ul Deepwell plate
############################################
WashBuffer2_450ul_plate2 = ctx.load_labware(
'kf_96_wellplate_2400ul', '10', 'Wash Buffer 2 Deepwell plate 2')
# Elution Deepwell plate
############################################
ElutionBuffer_50ul_plate = ctx.load_labware(
'kingfisher_std_96_wellplate_550ul', '6', 'Elution Buffer 50 ul STD plate')
####################################
# Load tip_racks
tips300 = [ctx.load_labware('opentrons_96_tiprack_300ul', slot, '200µl filter tiprack')
for slot in ['8']]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
WashBuffer1.reagent_reservoir = WashBuffer1_reservoir.wells()[0]
WashBuffer2.reagent_reservoir = WashBuffer2_reservoir.wells()[0]
ElutionBuffer.reagent_reservoir = reagent_res.rows()[0][0]
# columns in destination plates to be filled depending the number of samples
wb1plate1_destination = WashBuffer1_300ul_plate1.rows()[0][:num_cols]
wb1plate2_destination = WashBuffer1_300ul_plate2.rows()[0][:num_cols]
wb2plate1_destination = WashBuffer2_450ul_plate1.rows()[0][:num_cols]
wb2plate2_destination = WashBuffer2_450ul_plate2.rows()[0][:num_cols]
elutionbuffer_destination = ElutionBuffer_50ul_plate.rows()[0][:num_cols]
# pipette
m300 = ctx.load_instrument(
'p300_multi_gen2', 'right', tip_racks=tips300) # Load multi pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {m300: 0},
'maxes': {m300: len(tips300)*96}
}
############################################################################
# STEP 1 Filling with WashBuffer1 plate 1
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
wash_buffer_vol = [150, 150]
rinse = False # Only first time
########
# Wash buffer dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(wash_buffer_vol):
if (i == 0 and j == 0):
rinse = True #Rinse only first transfer
else:
rinse = False
move_vol_multichannel(m300, reagent = WashBuffer1, source = WashBuffer1.reagent_reservoir,
dest = wb1plate1_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 1, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = True)
m300.drop_tip(home_after=True)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2 Filling with WashBuffer1 plate 2
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
wash_buffer_vol = [150, 150]
rinse = False # Only first time
########
# Wash buffer dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(wash_buffer_vol):
if (i == 0 and j == 0):
rinse = True
else:
rinse = False
move_vol_multichannel(m300, reagent = WashBuffer1, source = WashBuffer1.reagent_reservoir,
dest = wb1plate2_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 1, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = True)
m300.drop_tip(home_after=True)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 3 Filling with WashBuffer2 plate 1
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
wash_buffer_vol = [150, 150, 150]
rinse = False # Only first time
########
# Wash buffer dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(wash_buffer_vol):
if (i == 0 and j == 0):
rinse = True
else:
rinse = False
move_vol_multichannel(m300, reagent = WashBuffer2, source = WashBuffer2.reagent_reservoir,
dest = wb2plate1_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 1, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = True)
m300.drop_tip(home_after=True)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 4 Filling with WashBuffer2 plate 2
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ethanol_vol = [150, 150, 150]
rinse = False # Only first time
########
# Ethanol dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(ethanol_vol):
if (i == 0 and j == 0):
rinse = True
else:
rinse = False
move_vol_multichannel(m300, reagent = WashBuffer2, source = WashBuffer2.reagent_reservoir,
dest = wb2plate2_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 1, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = True)
m300.drop_tip(home_after=True)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 5 Transfer Elution buffer
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
# Elution buffer
ElutionBuffer_vol = [50]
########
# Water or elution buffer
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for transfer_vol in ElutionBuffer_vol:
# Calculate pickup_height based on remaining volume and shape of container
[pickup_height, change_col] = calc_height(
ElutionBuffer, multi_well_rack_area, transfer_vol * 8)
ctx.comment(
'Aspirate from Reservoir column: ' + str(ElutionBuffer.col))
ctx.comment('Pickup height is ' + str(pickup_height))
move_vol_multichannel(m300, reagent = ElutionBuffer, source = ElutionBuffer.reagent_reservoir,
dest = elutionbuffer_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol_elutionbuffer, x_offset = x_offset,
pickup_height = pickup_height, rinse = False, disp_height = -2,
blow_out = True, touch_tip = False)
m300.drop_tip(home_after=True)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
from opentrons.drivers.rpi_drivers import gpio
for i in range(3):
gpio.set_rail_lights(False)
gpio.set_button_light(1, 0, 0)
time.sleep(0.3)
gpio.set_rail_lights(True)
gpio.set_button_light(0, 0, 1)
time.sleep(0.3)
gpio.set_button_light(0, 1, 0)
ctx.comment(
'Finished! \nMove deepwell plates to KingFisher extractor.')
ctx.comment('Used tips in total: ' + str(tip_track['counts'][m300]))
ctx.comment('Used racks in total: ' + str(tip_track['counts'][m300] / 96))
def run(ctx: protocol_api.ProtocolContext):
# Init protocol run
run = ProtocolRun(ctx)
run.comment("You are about to run %s samples\n STEPS:%s" %
(NUM_SAMPLES, steps),
add_hash=True)
run.pause(
"Are you sure the set up is correct? Check the desk before continue")
# Define stesp
run.add_step(
description="Transfer PK A6 - To AW_PLATE Single Slot1 -> Slot2") # 1
run.add_step(description="Transfer MS2 B6 - To AW_PLATE Single 1->2") # 4
run.add_step(description="Transfer Beats 3 - 2 Multi and mix") # 3
# execute avaliaible steps
run.init_steps(steps)
##################################
# Tube rack
tube_rack = ctx.load_labware('opentrons_24_tuberack_nest_1.5ml_screwcap',
4)
# Destination plate SLOT 2
if (ctx.is_simulating()):
aw_slot = ctx.load_labware(
'opentrons_96_aluminumblock_generic_pcr_strip_200ul', 5)
else:
aw_slot = ctx.load_labware('axygen_96_wellplate_2000ul', 5)
aw_wells = aw_slot.wells()[:NUM_SAMPLES]
aw_wells_multi = aw_slot.rows()[0][:num_cols]
# Magnetic Beads Pool
beads_slot = ctx.load_labware('nest_12_reservoir_15ml', 6)
beads_wells_multi = beads_slot.rows()[0][:num_cols]
# Mount pippets and set racks
# Tipracks20_multi
tips20 = ctx.load_labware('opentrons_96_tiprack_20ul', 7)
tips300_1 = ctx.load_labware('opentrons_96_filtertiprack_200ul', 8)
tips300_2 = ctx.load_labware('opentrons_96_filtertiprack_200ul', 9)
run.mount_right_pip('p20_single_gen2', tip_racks=[tips20], capacity=20)
run.mount_left_pip('p300_multi_gen2',
tip_racks=[tips300_1, tips300_2],
capacity=200,
multi=True)
############################################################################
# STEP 1: Transfer A6 - To AW_PLATE
############################################################################
if (run.next_step()):
run.set_pip("right") # single 20
volumen_move = 5
source = tube_rack.wells("A6")[0]
liquid = Reagent(
name='Proteinasa K',
num_wells=1, # change with num samples
flow_rate_aspirate=0.75, # Original 0.5
flow_rate_dispense=3, # Original 1
reagent_reservoir_volume=528,
h_cono=4,
v_fondo=4 * math.pi * 4**3 / 3)
run.pick_up()
for dest in aw_wells:
[pickup_height,
col_change] = run.calc_height(liquid, 4.12 * 4.12 * math.pi,
volumen_move)
run.move_volume(reagent=liquid,
source=source,
dest=dest,
vol=volumen_move,
air_gap_vol=1,
pickup_height=pickup_height,
disp_height=-10,
blow_out=True,
post_dispense=True,
post_dispense_vol=5)
run.drop_tip()
run.finish_step()
############################################################################
# STEP 2: Transfer B6 MS2 - To AW_PLATE
############################################################################
if (run.next_step()):
run.set_pip("right") # single 20
volumen_move = 5
source = tube_rack.wells("B6")[0]
liquid = Reagent(
name='MS2',
num_wells=1, # change with num samples
delay=0,
flow_rate_aspirate=3, # Original 0.5
flow_rate_dispense=3, # Original 1
flow_rate_aspirate_mix=15,
flow_rate_dispense_mix=25,
reagent_reservoir_volume=528,
h_cono=4,
v_fondo=4 * math.pi * 4**3 / 3)
run.pick_up()
for dest in aw_wells:
[pickup_height,
col_change] = run.calc_height(liquid, 4.12 * 4.12 * math.pi,
volumen_move)
run.move_volume(reagent=liquid,
source=source,
dest=dest,
vol=volumen_move,
air_gap_vol=1,
pickup_height=pickup_height,
disp_height=-10,
blow_out=True,
post_dispense=True,
post_dispense_vol=5)
run.drop_tip()
run.finish_step()
############################################################################
# STEP 3: Slot 3 -2 beats_PK AW
############################################################################
if (run.next_step()):
############################################################################
# Light flash end of program
run.set_pip("left") # p300 multi
volume = 275
beads = Reagent(name='Magnetic beads',
flow_rate_aspirate=0.5,
flow_rate_dispense=0.5,
flow_rate_dispense_mix=4,
flow_rate_aspirate_mix=4,
rinse=True,
delay=2,
reagent_reservoir_volume=30000,
num_wells=3,
h_cono=1.95,
v_fondo=695,
rinse_loops=3)
air_gap_vol = 5
disposal_height = -5
pickup_height = 1
beads.reagent_reservoir = beads_slot.rows()[0][0:3]
pool_area = 8.3 * 71.1
for destination in aw_wells_multi:
run.pick_up()
vol = 150
vol_min = 1000
[pickup_height, col_change] = run.calc_height(beads,
pool_area,
vol * 8,
extra_volume=vol_min)
run.move_volume(reagent=beads,
source=beads.reagent_reservoir[beads.col],
dest=destination,
vol=vol,
air_gap_vol=air_gap_vol,
pickup_height=pickup_height,
disp_height=disposal_height,
rinse=True,
blow_out=True)
run.change_tip()
vol = 125
[pickup_height, col_change] = run.calc_height(beads,
pool_area,
vol * 8,
extra_volume=vol_min)
run.move_volume(reagent=beads,
source=beads.reagent_reservoir[beads.col],
dest=destination,
vol=vol,
air_gap_vol=air_gap_vol,
pickup_height=pickup_height,
disp_height=disposal_height,
rinse=True,
blow_out=True)
run.custom_mix(beads,
location=destination,
vol=150,
rounds=3,
blow_out=True,
mix_height=0)
run.drop_tip()
run.finish_step()
run.log_steps_time()
run.blink()
for c in robot.commands():
ctx.comment(c)
ctx.comment('Finished! \nMove plate to PCR')
def run(ctx: protocol_api.ProtocolContext):
STEP = 0
STEPS = { # Dictionary with STEP activation, description and times
1: {
'Execute': True,
'description': 'Add Lysis buffer (' + str(volume_control) + 'ul)'
},
2: {
'Execute': True,
'description': 'Add samples (' + str(volume_sample) + 'ul)'
}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
if not ctx.is_simulating():
folder_path = '/var/lib/jupyter/notebooks/' + run_id
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/StationA_time_log.txt'
file_path2 = folder_path + '/StationA_tips_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self,
name,
flow_rate_aspirate,
flow_rate_dispense,
rinse,
reagent_reservoir_volume,
delay,
num_wells,
h_cono,
v_fondo,
tip_recycling='none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused = []
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
BUFFER = Reagent(name='TNA+Beads+Isopropanol',
flow_rate_aspirate=1,
flow_rate_dispense=1,
rinse=False,
delay=0,
reagent_reservoir_volume=50000,
num_wells=1,
h_cono=(v_cone_falcon * 3 / falcon_cross_section_area),
v_fondo=v_cone_falcon)
Samples = Reagent(
name='Samples',
flow_rate_aspirate=1,
flow_rate_dispense=1,
rinse=False,
delay=0,
reagent_reservoir_volume=700 * 24,
num_wells=24, # num_cols comes from available columns
h_cono=4,
v_fondo=4 * area_section_sample * diameter_sample * 0.5 / 3) # Sphere
BUFFER.vol_well = BUFFER.vol_well_original
Samples.vol_well = 700
##################
# Custom functions
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol,
x_offset, pickup_height, rinse, disp_height,
blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet,
reagent,
location=source,
vol=vol,
rounds=2,
blow_out=True,
mix_height=0,
x_offset=x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x=x_offset[0]))
pipet.aspirate(vol, s) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol,
source.top(z=-2),
rate=reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z=disp_height).move(Point(x=x_offset[1]))
pipet.dispense(vol + air_gap_vol,
drop,
rate=reagent.flow_rate_dispense) # dispense all
ctx.delay(
seconds=reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z=-2))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-5)
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
x_offset,
source_height=3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
pipet.dispense(vol,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
pipet.dispense(1,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
def calc_height(reagent,
cross_section_area,
aspirate_volume,
min_height=0.5):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
def generate_source_table(source):
'''
Concatenate the wells frome the different origin racks
'''
for rack_number in range(len(source)):
if rack_number == 0:
s = source[rack_number].wells()
else:
s = s + source[rack_number].wells()
return s
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) +
'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
####################################
# load labware and modules
####################################
# Load Sample racks
if NUM_SAMPLES < 96:
rack_num = math.ceil(NUM_SAMPLES / 24)
ctx.comment('Used source racks are ' + str(rack_num))
samples_last_rack = NUM_SAMPLES - rack_num * 24
else:
rack_num = 4
source_racks = [
ctx.load_labware('opentrons_24_tuberack_generic_2ml_screwcap', slot,
'source tuberack with screwcap' + str(i + 1))
for i, slot in enumerate(['4', '1', '6', '3'][:rack_num])
]
##################################
# Destination plate
dest_plate = ctx.load_labware('abgene_96_wellplate_800ul', '5',
'ABGENE 96 Well Plate 800 µL')
############################################
# tempdeck
#tempdeck = ctx.load_module('tempdeck', '1')
#tempdeck.set_temperature(temperature)
##################################
# Cooled reagents in tempdeck
#reagents = tempdeck.load_labware(
#'opentrons_24_aluminumblock_generic_2ml_screwcap',
#'cooled reagent tubes')
reagents = ctx.load_labware('opentrons_6_tuberack_falcon_50ml_conical',
'7', 'Lysis buffer tuberack in Falcon tube')
####################################
# Load tip_racks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot,
'20µl filter tiprack') for slot in ['11']
]
tips1000 = [
ctx.load_labware('opentrons_96_filtertiprack_1000ul', slot,
'1000µl filter tiprack') for slot in ['10']
]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
BUFFER.reagent_reservoir = reagents.wells()[0]
# setup samples and destinations
sample_sources_full = generate_source_table(source_racks)
sample_sources = sample_sources_full[:NUM_SAMPLES]
destinations = dest_plate.wells()[:NUM_SAMPLES]
p20 = ctx.load_instrument('p20_single_gen2',
mount='right',
tip_racks=tips20)
p1000 = ctx.load_instrument('p1000_single_gen2',
'left',
tip_racks=tips1000) # load P1000 pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {
p20: 0,
p1000: 0
},
'maxes': {
p20: len(tips20) * 96,
p1000: len(tips1000) * 96
}
}
############################################################################
# STEP 1: Add TNA
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
# Transfer parameters
start = datetime.now()
if not p1000.hw_pipette['has_tip']:
pick_up(p1000)
for d in destinations:
# Calculate pickup_height based on remaining volume and shape of container
[pickup_height,
change_col] = calc_height(BUFFER, falcon_cross_section_area,
volume_control)
move_vol_multichannel(p1000,
reagent=BUFFER,
source=BUFFER.reagent_reservoir,
dest=d,
vol=volume_control,
air_gap_vol=air_gap_vol_ci,
x_offset=x_offset,
pickup_height=pickup_height,
rinse=BUFFER.rinse,
disp_height=height_control,
blow_out=True,
touch_tip=True)
# Mix the sample AFTER dispensing using 15µl of volume
#custom_mix(p20, reagent = Control_I, location = d, vol = 15, rounds = 4, blow_out = True, mix_height = 15)
#Do not drop tip as it is not contaminated
#p1000.drop_tip()
#tip_track['counts'][p20]+=1
#Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: Add Samples
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
# Transfer parameters
start = datetime.now()
for s, d in zip(sample_sources, destinations):
if not p1000.hw_pipette['has_tip']:
pick_up(p1000)
# Mix the sample BEFORE dispensing
#custom_mix(p1000, reagent = Samples, location = s, vol = volume_sample, rounds = 2, blow_out = True, mix_height = 15)
move_vol_multichannel(p1000,
reagent=Samples,
source=s,
dest=d,
vol=volume_sample,
air_gap_vol=air_gap_vol_sample,
x_offset=x_offset,
pickup_height=1,
rinse=Samples.rinse,
disp_height=-10,
blow_out=True,
touch_tip=True)
# Mix the sample AFTER dispensing using 15µl of volume
custom_mix(p1000,
reagent=Samples,
location=d,
vol=800,
rounds=2,
blow_out=False,
mix_height=10)
p1000.drop_tip()
tip_track['counts'][p1000] += 1
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
with open(file_path2, 'w') as f2:
f2.write('pipette\ttip_count\n')
for key in tip_track['counts'].keys():
row = str(key)
f.write(str(key) + '\t' + format(tip_track['counts'][key]))
f2.close()
############################################################################
# Light flash end of program
from opentrons.drivers.rpi_drivers import gpio
#if not ctx.is_simulating():
#os.system('mpg123 -f -14000 /var/lib/jupyter/notebooks/lionking.mp3')
for i in range(3):
gpio.set_rail_lights(False)
gpio.set_button_light(1, 0, 0)
time.sleep(0.3)
gpio.set_rail_lights(True)
gpio.set_button_light(0, 0, 1)
time.sleep(0.3)
gpio.set_button_light(0, 1, 0)
ctx.comment(
'Finished! \nMove deepwell plate (slot 5) to Station B for extraction protocol'
)
ctx.comment('Used p1000 tips in total: ' + str(tip_track['counts'][p1000]))
ctx.comment('Used p1000 racks in total: ' +
str(tip_track['counts'][p1000] / 96))
ctx.comment('Used p20 tips in total: ' + str(tip_track['counts'][p20]))
ctx.comment('Used p20 racks in total: ' +
str(tip_track['counts'][p20] / 96))
def run(ctx: protocol_api.ProtocolContext):
STEP = 0
STEPS = { # Dictionary with STEP activation, description and times
1: {
'Execute':
True,
'description':
'Mezclar y dispensar muestras (' + str(VOLUME_SAMPLE) + 'ul)'
}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
if not ctx.is_simulating():
folder_path = '/var/lib/jupyter/notebooks/' + run_id
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/StationA_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
delay):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.delay = delay
# Reagents and their characteristics
Samples = Reagent(name='Samples',
flow_rate_aspirate=25,
flow_rate_dispense=100,
rinse=False,
delay=0)
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('CONTROLES: ' + str(NUM_CONTROL_SPACES))
ctx.comment('MUESTRAS: ' + str(NUM_REAL_SAMPLES))
ctx.comment('###############################################')
ctx.comment(' ')
##################
# Custom functions
def move_vol_multichannel(pipet,
reagent,
source,
dest,
vol,
air_gap_vol,
x_offset,
pickup_height,
rinse,
disp_height,
blow_out,
touch_tip,
shakes=0,
shake_v_offset=-45):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet,
reagent,
location=source,
vol=vol,
rounds=2,
blow_out=True,
mix_height=0,
x_offset=x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x=x_offset[0]))
pipet.aspirate(vol, s,
rate=reagent.flow_rate_aspirate) # aspirate liquid
if shakes > 0:
shake_pipet(pipet, v_offset=shake_v_offset)
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol,
source.top(z=-2),
rate=reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z=disp_height).move(Point(x=x_offset[1]))
pipet.dispense(vol + air_gap_vol,
drop,
rate=reagent.flow_rate_dispense) # dispense all
ctx.delay(
seconds=reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z=disp_height))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-10)
if air_gap_vol != 0:
pipet.air_gap(air_gap_vol, height=disp_height) #air gap
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
x_offset,
source_height=5):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height <= 0:
mix_height = 3
pipet.aspirate(1,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
pipet.dispense(vol,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
pipet.dispense(1,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
def generate_source_table(source):
'''
Concatenate the wells frome the different origin racks
'''
num_cols = math.ceil(num_samples / 8)
s = []
for i in range(num_cols):
if i < 6:
s += source[0].columns()[i] + source[1].columns()[i]
else:
s += source[2].columns()[i - 6] + source[3].columns()[i - 6]
return s
def shake_pipet(pipet, rounds=2, speed=100, v_offset=0):
ctx.comment("Shaking " + str(rounds) + " rounds.")
for i in range(rounds):
pipet.touch_tip(speed=speed, radius=0.1, v_offset=v_offset)
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) +
'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
def run_quiet_process(command):
subprocess.check_output('{} &> /dev/null'.format(command), shell=True)
def play_sound(filename):
print('Speaker')
print('Next\t--> CTRL-C')
try:
run_quiet_process('mpg123 {}'.format(path_sounds + filename +
'.mp3'))
run_quiet_process('mpg123 {}'.format(path_sounds + sonido_defecto))
run_quiet_process('mpg123 {}'.format(path_sounds + filename +
'.mp3'))
except KeyboardInterrupt:
pass
print()
def start_run():
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Empezando protocolo')
if PHOTOSENSITIVE == False:
ctx._hw_manager.hardware.set_lights(button=True, rails=True)
else:
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
now = datetime.now()
# dd/mm/YY H:M:S
start_time = now.strftime("%Y/%m/%d %H:%M:%S")
return start_time
def finish_run(switch_off_lights=False):
ctx.comment('###############################################')
ctx.comment('Protocolo finalizado')
ctx.comment(' ')
#Set light color to blue
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
now = datetime.now()
# dd/mm/YY H:M:S
finish_time = now.strftime("%Y/%m/%d %H:%M:%S")
if PHOTOSENSITIVE == False:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button=False, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=True)
time.sleep(0.3)
else:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button=False, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
time.sleep(0.3)
if switch_off_lights:
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
used_tips = tip_track['num_refills'][p1000] * 96 * len(
p1000.tip_racks) + tip_track['counts'][p1000]
ctx.comment('Puntas de 1000 ul utilizadas: ' + str(used_tips) + ' (' +
str(round(used_tips / 96, 2)) + ' caja(s))')
ctx.comment('###############################################')
if not ctx.is_simulating():
for i in range(SOUND_NUM_PLAYS):
if i > 0:
time.sleep(60)
play_sound('finished_process_esp')
return finish_time
####################################
# load labware and modules
####################################
# Load Sample racks
if num_samples <= 48:
rack_num = 2
ctx.comment('Used source racks are ' + str(rack_num))
else:
rack_num = 4
#source_racks = [ctx.load_labware(
# 'pcr_24_wellplate_13200ul', slot,
# 'source tuberack with snapcap' + str(i + 1)) for i, slot in enumerate(['4', '1', '5', '2'][:rack_num])
#]
source_racks = [
ctx.load_labware('vitrobiocomma_24_tuberack_15000ul_1', '4',
'samples grid 1'),
ctx.load_labware('vitrobiocomma_24_tuberack_15000ul_2', '1',
'samples grid 2'),
ctx.load_labware('vitrobiocomma_24_tuberack_15000ul_3', '5',
'samples grid 3'),
ctx.load_labware('vitrobiocomma_24_tuberack_15000ul_4', '2',
'samples grid 4')
]
##################################
# Destination plate
dest_plate = ctx.load_labware('nest_96_wellplate_2ml_deep', '6',
'NEST 96 Deepwell Plate 2mL')
####################################
# Load tip_racks
tips1000 = [
ctx.load_labware(
'opentrons_96_filtertiprack_1000ul' if OPENTRONS_TIPS else
'geb_96_tiprack_1000ul', slot, '1000µl filter tiprack')
for slot in ['8']
]
################################################################################
# setup samples and destinations
sample_sources_full = generate_source_table(source_racks)
sample_sources = sample_sources_full[NUM_CONTROL_SPACES:num_samples]
destinations = dest_plate.wells()[NUM_CONTROL_SPACES:num_samples]
p1000 = ctx.load_instrument('p1000_single_gen2',
'left',
tip_racks=tips1000) # load P1000 pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {
p1000: 0
},
'maxes': {
p1000: 96 * len(p1000.tip_racks)
}, #96 tips per tiprack * number or tipracks in the layout
'num_refills': {
p1000: 0
},
'tips': {
p1000: [tip for rack in tips1000 for tip in rack.rows()[0]]
}
}
start_run()
############################################################################
# STEP 1: MIX AND MOVE SAMPLES
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
start = datetime.now()
for s, d in zip(sample_sources, destinations):
if not p1000.hw_pipette['has_tip']:
pick_up(p1000)
# Mix the sample BEFORE dispensing
if NUM_MIXES > 0:
custom_mix(p1000,
reagent=Samples,
location=s,
vol=volume_mix,
rounds=NUM_MIXES,
blow_out=True,
mix_height=5,
x_offset=x_offset)
move_vol_multichannel(p1000,
reagent=Samples,
source=s,
dest=d,
vol=VOLUME_SAMPLE,
air_gap_vol=air_gap_vol_sample,
x_offset=x_offset,
pickup_height=8,
rinse=Samples.rinse,
disp_height=-10,
blow_out=True,
touch_tip=False,
shakes=SAMPLE_SAKES)
p1000.drop_tip(home_after=False)
tip_track['counts'][p1000] += 1
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
# from opentrons.drivers.rpi_drivers import gpio
finish_run(switch_off_lights)
def run(ctx: protocol_api.ProtocolContext):
from opentrons.drivers.rpi_drivers import gpio
ctx.comment('Actual used columns: ' + str(num_cols))
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {
'Execute': True,
'description': 'Add MS2'
},
2: {
'Execute': True,
'description': 'Transfer beads'
}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
folder_path = '/var/lib/jupyter/notebooks'
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/Station_KB_sample_prep_viral_path2_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self,
name,
flow_rate_aspirate,
flow_rate_dispense,
rinse,
reagent_reservoir_volume,
delay,
num_wells,
h_cono,
v_fondo,
tip_recycling='none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused = []
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
Sample = Reagent(name='Sample',
flow_rate_aspirate=1,
flow_rate_dispense=1,
rinse=True,
delay=0,
reagent_reservoir_volume=460 * 96,
num_wells=96,
h_cono=1.95,
v_fondo=35)
Beads = Reagent(name='Magnetic beads and binding solution',
flow_rate_aspirate=0.75,
flow_rate_dispense=0.75,
rinse=True,
num_wells=math.ceil(NUM_SAMPLES / 32),
delay=2,
reagent_reservoir_volume=550 * 8 * num_cols * 1.1,
h_cono=1.95,
v_fondo=695) # Prismatic
MS = Reagent(
name='MS2',
flow_rate_aspirate=1,
flow_rate_dispense=1,
rinse=False,
reagent_reservoir_volume=total_MS_volume,
num_wells=8,
delay=0,
h_cono=h_cone,
v_fondo=volume_cone # V cono
) # Prismatic)
Sample.vol_well = Sample.reagent_reservoir_volume
Beads.vol_well = Beads.vol_well_original
MS.vol_well = MS.reagent_reservoir_volume
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol,
x_offset, pickup_height, rinse, disp_height,
blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet,
reagent,
location=source,
vol=vol,
rounds=2,
blow_out=True,
mix_height=0,
x_offset=x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x=x_offset[0]))
pipet.aspirate(vol, s,
rate=reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol,
source.top(z=-2),
rate=reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z=disp_height).move(Point(x=x_offset[1]))
pipet.dispense(vol + air_gap_vol,
drop,
rate=reagent.flow_rate_dispense) # dispense all
ctx.delay(
seconds=reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z=-2))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-5)
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
x_offset,
source_height=3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
pipet.dispense(vol,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
pipet.dispense(1,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
def calc_height(reagent,
cross_section_area,
aspirate_volume,
min_height=0.5,
extra_volume=50):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume + extra_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
def divide_destinations(l, n):
# Divide the list of destinations in size n lists.
for i in range(0, len(l), n):
yield l[i:i + n]
####################################
# load labware and modules
# 12 well rack
reagent_res = ctx.load_labware('perkinelmer_12_reservoir_21000ul', '2',
'Reagent deepwell plate')
##################################
# Sample prep plate - final plate, goes to Kingfisher
sample_plate = ctx.load_labware('kf_96_wellplate_2400ul', '1',
'KF 96 Well 2400ul elution plate')
############################################
# tempdeck
tempdeck = ctx.load_module('tempdeck', '4')
tempdeck.set_temperature(temperature)
##################################
# MS plate - plate with a column containing the internal control MS
ms_plate = tempdeck.load_labware('vwr_96_wellplate_200ul_alum_opentrons',
'pcr plate with MS control')
####################################
# load labware and modules
# 24 well rack aluminium opentrons
#tuberack = ctx.load_labware(
# 'opentrons_24_aluminumblock_generic_2ml_screwcap', '3',
# 'Bloque Aluminio opentrons 24 Screwcaps')
##################################
# Load Tipracks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['6']
]
tips200 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['3']
]
# pipettes. P1000 currently deactivated
m300 = ctx.load_instrument('p300_multi_gen2', 'right',
tip_racks=tips200) # Load multi pipette
m20 = ctx.load_instrument('p20_multi_gen2', 'left', tip_racks=tips20)
#p20 = ctx.load_instrument(
# 'p20_single_gen2', 'left', tip_racks=tips20) # load P1000 pipette
tip_track = {
'counts': {
m300: 0,
m20: 0
},
'maxes': {
m300: len(tips200) * 96,
m20: len(tips20) * 96
}
}
# Divide destination wells in small groups for P300 pipette
# Declare which reagents are in each reservoir as well as deepwell and elution plate
#destinations = list(divide_destinations(sample_plate.wells()[:NUM_SAMPLES], size_transfer))
Beads.reagent_reservoir = reagent_res.rows(
)[0][:Beads.num_wells] # 1 row, 4 columns (first ones)
work_destinations = sample_plate.wells()[:NUM_SAMPLES]
work_destinations_cols = sample_plate.rows()[0][:num_cols]
ms_origins = ms_plate.rows()[0][0] # 1 row, 1 columns
############################################################################
# STEP 1: Transfer MS
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('ms_wells')
#Loop over defined wells
for d in work_destinations_cols:
m20.pick_up_tip()
#Source samples
move_vol_multichannel(m20,
reagent=MS,
source=ms_origins,
dest=d,
vol=MS_vol,
air_gap_vol=air_gap_vol_MS,
x_offset=x_offset,
pickup_height=0.2,
disp_height=-35,
rinse=False,
blow_out=True,
touch_tip=True)
m20.drop_tip()
tip_track['counts'][m20] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# STEP 2: TRANSFER BEADS
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
# Transfer parameters
start = datetime.now()
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
beads_transfer_vol = [150, 150, 150,
100] # 4 rounds of different volumes
rinse = True
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
m300.pick_up_tip()
for j, transfer_vol in enumerate(beads_transfer_vol):
# Calculate pickup_height based on remaining volume and shape of container
[pickup_height, change_col] = calc_height(Beads,
multi_well_rack_area,
transfer_vol * 8,
min_height=1)
if change_col == True: # If we switch column because there is not enough volume left in current reservoir column we mix new column
ctx.comment('Mixing new reservoir column: ' +
str(Beads.col))
custom_mix(m300,
Beads,
Beads.reagent_reservoir[Beads.col],
vol=170,
rounds=10,
blow_out=False,
mix_height=0,
x_offset=x_offset)
ctx.comment('Aspirate from reservoir column: ' +
str(Beads.col))
ctx.comment('Pickup height is ' + str(pickup_height))
if j != 0:
rinse = False
move_vol_multichannel(
m300,
reagent=Beads,
source=Beads.reagent_reservoir[Beads.col],
dest=work_destinations_cols[i],
vol=transfer_vol,
air_gap_vol=air_gap_vol,
x_offset=x_offset,
pickup_height=pickup_height,
disp_height=-2,
rinse=rinse,
blow_out=False,
touch_tip=False)
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
gpio.set_rail_lights(False)
time.sleep(2)
#os.system('mpg123 -f -8000 /var/lib/jupyter/notebooks/toreador.mp3 &')
for i in range(3):
gpio.set_rail_lights(False)
gpio.set_button_light(1, 0, 0)
time.sleep(0.3)
gpio.set_rail_lights(True)
gpio.set_button_light(0, 0, 1)
time.sleep(0.3)
gpio.set_button_light(0, 1, 0)
ctx.comment('Finished! \nMove plate to KingFisher')
def run(ctx: protocol_api.ProtocolContext):
import os
ctx.comment('Actual used columns: ' + str(num_cols))
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {'Execute': False, 'description': 'Make MMIX'},
2: {'Execute': False, 'description': 'Transfer MMIX with P300'},
3: {'Execute': True, 'description': 'Transfer MMIX with P20'},
4: {'Execute': True, 'description': 'Clean up NC and PC wells'},
5: {'Execute': True, 'description': 'Transfer elution'},
6: {'Execute': True, 'description': 'Transfer PC'},
7: {'Execute': True, 'description': 'Transfer NC'}
}
if STEPS[2]['Execute']==True:
STEPS[3]['Execute']=False # just to make sure if P300 is being used for the MMIX, do not load P20 single
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
folder_path = '/var/lib/jupyter/notebooks/'+str(run_id)
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/KC_qPCR_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
reagent_reservoir_volume, delay, num_wells, h_cono, v_fondo,
tip_recycling = 'none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused=[]
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
MMIX = Reagent(name = 'Master Mix',
rinse = False,
flow_rate_aspirate = 2,
flow_rate_dispense = 4,
reagent_reservoir_volume = $MMIX_total_volume, # volume_mmix_available,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
NC = Reagent(name = 'Negative control',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000, # volume_mmix_available,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
PC = Reagent(name = 'Positive control',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000, # volume_mmix_available,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
Elution = Reagent(name='Elution',
rinse=False,
flow_rate_aspirate = 1,
flow_rate_dispense = 2,
reagent_reservoir_volume=50,
delay=1,
num_wells=num_cols, # num_cols comes from available columns
h_cono=0,
v_fondo=0
)
tackpath = Reagent(name = 'MMIX_multiplex_tackpath',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
covid_assay = Reagent(name = 'Covid19_assay',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
mmix_water = Reagent(name = 'nuclease_free_water',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
component4 = Reagent(name = 'component4',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
component5 = Reagent(name = 'component5',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
component6 = Reagent(name = 'component6',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
component7 = Reagent(name = 'component7',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
component8 = Reagent(name = 'component8',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 1000,
num_wells = 1, #change with num samples
delay = 0,
h_cono = h_cone,
v_fondo = volume_cone # V cono
)
MMIX.vol_well = MMIX.vol_well_original
PC.vol_well = PC.vol_well_original
NC.vol_well = NC.vol_well_original
Elution.vol_well = Elution.vol_well_original
tackpath.vol_well = tackpath.vol_well_original
covid_assay.vol_well = covid_assay.vol_well_original
mmix_water.vol_well = mmix_water.vol_well_original
component4.vol_well = component4.vol_well_original
component5.vol_well = component5.vol_well_original
component6.vol_well = component6.vol_well_original
component7.vol_well = component7.vol_well_original
component8.vol_well = component8.vol_well_original
################## Assign class type reactives to a summary
MMIX_components=[tackpath, covid_assay, mmix_water, component4, component5, component6, component7, component8]
MMIX_components=MMIX_components[:len(MMIX_make[mmix_selection])]
##################
##################
# Custom functions
def divide_volume(volume,max_vol):
num_transfers=math.ceil(volume/max_vol)
vol_roundup=math.ceil(volume/num_transfers)
last_vol = volume - vol_roundup*(num_transfers-1)
vol_list = [vol_roundup for v in range(1,num_transfers)]
vol_list.append(last_vol)
return vol_list
def divide_destinations(l, n): # Divide the list of destinations in size n lists.
a=[]
for i in range(0, len(l), n):
a.append( l[i:i + n])
return a
def distribute_custom(pipette, reagent, volume, src, dest, waste_pool, pickup_height, extra_dispensal, disp_height=0):
# Custom distribute function that allows for blow_out in different location and adjustement of touch_tip
air_gap=10
pipette.aspirate((len(dest) * volume) +
extra_dispensal, src.bottom(pickup_height), rate = reagent.flow_rate_aspirate)
pipette.touch_tip(speed=20, v_offset=-5)
pipette.move_to(src.top(z=5))
pipette.aspirate(air_gap, rate = reagent.flow_rate_aspirate) # air gap
for d in dest:
pipette.dispense(air_gap, d.top(), rate = reagent.flow_rate_dispense)
drop = d.top(z = disp_height)
pipette.dispense(volume, drop, rate = reagent.flow_rate_dispense)
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
pipette.move_to(d.top(z=5))
pipette.aspirate(air_gap,d.top(z=5), rate = reagent.flow_rate_aspirate) # air gap
try:
pipette.blow_out(waste_pool.wells()[0].bottom(pickup_height + 3))
except:
pipette.blow_out(waste_pool.bottom(pickup_height + 3))
return (len(dest) * volume)
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip,
post_dispense=False, post_dispense_vol=20,
post_airgap=False, post_airgap_vol=10):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = 2, blow_out = True, mix_height = 0,
x_offset = x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s, rate = reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z = -2))
if post_dispense == True:
pipet.dispense(post_dispense_vol, dest.top(z = -2))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -5, radius = 0.9)
if post_airgap == True:
pipet.aspirate(post_airgap_vol, dest.top(z = 5), rate = 2)
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
x_offset, source_height = 3, post_airgap=False, post_airgap_vol=10,
post_dispense=False, post_dispense_vol=20,touch_tip=False):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
pipet.dispense(vol, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
pipet.dispense(1, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
if post_dispense == True:
pipet.dispense(post_dispense_vol, location.top(z = -2))
if post_airgap == True:
pipet.dispense(post_airgap_vol, location.top(z = 5))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -5, radius = 0.9)
def calc_height(reagent, cross_section_area, aspirate_volume, min_height = 0.3, extra_volume = 30):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume + extra_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
############################################
# tempdeck
tempdeck = ctx.load_module('tempdeck', '1') #temdeck for qpcr samples
tempdeck.set_temperature(temperature)
tempdeck_two = ctx.load_module('tempdeck', '4') #tempdeck for MMIX, PC and NC
tempdeck_two.set_temperature(temperature)
####################################
# load labware and modules
# 24 well rack
tuberack = tempdeck_two.load_labware(
'opentrons_24_aluminumblock_generic_2ml_screwcap',
'Bloque Aluminio opentrons 24 screwcaps 2000 µL ')
##################################
# qPCR plate - final plate, goes to PCR
qpcr_plate = tempdeck.load_labware(
'abi_fast_qpcr_96_alum_opentrons_100ul',
'chilled qPCR final plate')
##################################
# waste reservoir
waste_reservoir = ctx.load_labware(
'nest_1_reservoir_195ml', '9', 'waste reservoir')
waste = waste_reservoir.wells()[0] # referenced as reservoir
##################################
# Sample plate - comes from B
source_plate = ctx.load_labware(
"kingfisher_std_96_wellplate_550ul", '2',
'chilled KF plate with elutions (alum opentrons)')
samples = source_plate.wells()[:NUM_SAMPLES]
##################################
# Load Tipracks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['5']
]
tips200 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['6']
]
# pipettes
m20 = ctx.load_instrument(
'p20_multi_gen2', mount='left', tip_racks=tips20)
if STEPS[2]['Execute']==True:
p300 = ctx.load_instrument(
'p300_single_gen2', mount='right', tip_racks=tips200)
# used tips counter
tip_track = {
'counts': {p300: 0, m20: 0},
'maxes': {p300: len(tips200) * 96, m20: len(tips20)*96}
}
else:
tips20mmix = [ ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['8'] ]
p20 = ctx.load_instrument(
'p20_single_gen2', mount='right', tip_racks=tips20mmix)
# used tips counter
tip_track = {
'counts': {p20: 0, m20: 0},
'maxes': {p20: len(tips200) * 96, m20: len(tips20)*96}
}
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
MMIX.reagent_reservoir = tuberack.rows()[0][:MMIX.num_wells] # 1 row, 2 columns (first ones)
MMIX_components_location=tuberack.wells()[MMIX_make_location:(MMIX_make_location + len(MMIX_make[mmix_selection]))]
ctx.comment('Wells in: '+ str(tuberack.rows()[0][:MMIX.num_wells]) + ' element: '+str(MMIX.reagent_reservoir[MMIX.col]))
PC.reagent_reservoir = tuberack.rows()[1][:1]
NC.reagent_reservoir = tuberack.rows()[2][:1]
# setup up sample sources and destinations
samples = source_plate.wells()[:NUM_SAMPLES]
samples_multi = source_plate.rows()[0][:num_cols]
pcr_wells = qpcr_plate.wells()[:(NUM_SAMPLES)]
pcr_wells_multi = qpcr_plate.rows()[0][:num_cols]
pc_well_old = source_plate.wells()[(NUM_SAMPLES-2):(NUM_SAMPLES-1)][0]
nc_well_old = source_plate.wells()[(NUM_SAMPLES-1):(NUM_SAMPLES)][0]
pc_well = qpcr_plate.wells()[(NUM_SAMPLES-2):(NUM_SAMPLES-1)][0]
nc_well = qpcr_plate.wells()[(NUM_SAMPLES-1):(NUM_SAMPLES)][0]
# Divide destination wells in small groups for P300 pipette
dests = list(divide_destinations(pcr_wells, size_transfer))
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
if not pip.hw_pipette['has_tip']:
pip.pick_up_tip()
##########
############################################################################
# STEP 1: Make Master MIX
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
# Check if among the pipettes, p300_single is installed
used_vol=[]
ctx.comment('#######################################################')
ctx.comment('Selected MMIX: '+MMIX_available[mmix_selection])
ctx.comment('#######################################################')
for i,[source, vol] in enumerate(zip(MMIX_components_location, MMIX_make[mmix_selection])):
pick_up(p300)
ctx.comment('#######################################################')
ctx.comment('Add component: '+MMIX_components[i].name)
ctx.comment('#######################################################')
if (vol + air_gap_vol) > pipette_allowed_capacity: # because 200ul is the maximum volume of the tip we will choose 180
# calculate what volume should be transferred in each step
vol_list=divide_volume(vol, pipette_allowed_capacity)
for vol in vol_list:
move_vol_multichannel(p300, reagent=MMIX_components[i], source=source, dest=MMIX.reagent_reservoir[0],
vol=vol, air_gap_vol=air_gap_vol, x_offset = x_offset,pickup_height=1, # should be changed with picku_up_height calculation
rinse=False, disp_height=-10,blow_out=True, touch_tip=True)
else:
move_vol_multichannel(p300, reagent=MMIX_components[i], source=source, dest=MMIX.reagent_reservoir[0],
vol=vol, air_gap_vol=air_gap_vol, x_offset=x_offset, pickup_height=1, # should be changed with picku_up_height calculation
rinse=False, disp_height=-10,blow_out=True, touch_tip=True)
if i+1<len(MMIX_components):
p300.drop_tip()
else:
ctx.comment('#######################################################')
ctx.comment('Final mix')
ctx.comment('#######################################################')
custom_mix(p300, reagent = MMIX, location = MMIX.reagent_reservoir[0], vol = 180, rounds = 5,
blow_out = True, mix_height = 2, x_offset = x_offset)
p300.drop_tip()
tip_track['counts'][p300]+=1
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: Transfer Master MIX with P300
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
p300.pick_up_tip()
for dest in pcr_wells:
[pickup_height, col_change] = calc_height(MMIX, area_section_screwcap, volume_mmix)
move_vol_multichannel(p300, reagent = MMIX, source = MMIX.reagent_reservoir[MMIX.col],
dest = dest, vol = volume_mmix, air_gap_vol = air_gap_mmix, x_offset = x_offset,
pickup_height = pickup_height, disp_height = -10, rinse = False,
blow_out=True, touch_tip=False)
#used_vol.append(used_vol_temp)
p300.drop_tip()
tip_track['counts'][p300]+=1
#MMIX.unused_two = MMIX.vol_well
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 3: Transfer Master MIX with P20
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
p20.pick_up_tip()
for dest in pcr_wells:
[pickup_height, col_change] = calc_height(MMIX, area_section_screwcap, volume_mmix)
move_vol_multichannel(p20, reagent = MMIX, source = MMIX.reagent_reservoir[MMIX.col],
dest = dest, vol = volume_mmix, air_gap_vol = 0, x_offset = x_offset,
pickup_height = pickup_height, disp_height = -10, rinse = False,
blow_out=True, touch_tip=True)
#used_vol.append(used_vol_temp)
p20.drop_tip()
tip_track['counts'][p20]+=1
#MMIX.unused_two = MMIX.vol_well
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
ctx.pause('Put samples please')
############################################################################
# STEP 3: Clean up PC and NC well
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
clean_up_wells=[pc_well_old,nc_well_old]
p20.pick_up_tip()
for src in clean_up_wells:
for i in range(3):
move_vol_multichannel(p20, reagent = PC, source = src,
dest = waste, vol = 20, air_gap_vol = 0, x_offset = [0,0],
pickup_height = 0.2, disp_height = 5, rinse = False,
blow_out=True, touch_tip=False,post_airgap=False, post_airgap_vol=0)
p20.drop_tip()
tip_track['counts'][p20]+=1
#MMIX.unused_two = MMIX.vol_well
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 4: TRANSFER Samples
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('pcr_wells')
#Loop over defined wells
for s, d in zip(samples_multi, pcr_wells_multi):
m20.pick_up_tip()
#Source samples
move_vol_multichannel(m20, reagent = Elution, source = s, dest = d,
vol = volume_sample, air_gap_vol = air_gap_sample, x_offset = x_offset,
pickup_height = 0.3, disp_height = -10, rinse = False,
blow_out=True, touch_tip=False, post_airgap=False)
# Mixing
custom_mix(m20, Elution, d, vol=5, rounds=2, blow_out=True,
mix_height=6, post_dispense=True, source_height=0.5, x_offset=[0,0],touch_tip=True)
m20.drop_tip()
tip_track['counts'][m20]+=8
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 5: Transfer PC with P20
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
p20.pick_up_tip()
#[pickup_height, col_change] = calc_height(PC, area_section_screwcap, volume_mmix)
move_vol_multichannel(p20, reagent = PC, source = PC.reagent_reservoir[PC.col],
dest = pc_well, vol = volume_pc, air_gap_vol = 0, x_offset = x_offset,
pickup_height = 0.2, disp_height = -10, rinse = False,
blow_out=True, touch_tip=True)
p20.drop_tip(home_after=False)
tip_track['counts'][p20]+=1
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 6: Transfer NC with P20
############################################################################
ctx._hw_manager.hardware.set_lights(rails=False) # set lights off when using MMIX
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
p20.pick_up_tip()
move_vol_multichannel(p20, reagent = NC, source = NC.reagent_reservoir[NC.col],
dest = nc_well, vol = volume_nc, air_gap_vol = 0, x_offset = x_offset,
pickup_height = 0.2, disp_height = -10, rinse = False,
blow_out=True, touch_tip=True)
p20.drop_tip(home_after=False)
tip_track['counts'][p20]+=1
#MMIX.unused_two = MMIX.vol_well
end = datetime.now()
time_taken = (end - start)
ctx.comment('#######################################################')
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
ctx.comment('#######################################################')
STEPS[STEP]['Time:'] = str(time_taken)
tempdeck.deactivate()
tempdeck_two.deactivate()
############################################################################
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
ctx.home()
time.sleep(2)
import os
#os.system('mpg123 -f -8000 /etc/audio/speaker-test.mp3 &')
'''if STEPS[1]['Execute'] == True:
total_used_vol = np.sum(used_vol)
total_needed_volume = total_used_vol
ctx.comment('Total Master Mix used volume is: ' + str(total_used_vol) + '\u03BCl.')
ctx.comment('Needed Master Mix volume is ' +
str(total_needed_volume + extra_dispensal*len(dests)) +'\u03BCl')
ctx.comment('Used Master Mix volumes per run are: ' + str(used_vol) + '\u03BCl.')
ctx.comment('Master Mix Volume remaining in tubes is: ' +
format(np.sum(MMIX.unused)+extra_dispensal*len(dests)+MMIX.vol_well) + '\u03BCl.')
ctx.comment('200 ul Used tips in total: ' + str(tip_track['counts'][p300]))
ctx.comment('200 ul Used racks in total: ' + str(tip_track['counts'][p300] / 96))'''
if (STEPS[3]['Execute'] == True & STEPS[4]['Execute'] == True):
ctx.comment('20 ul Used tips in total: ' + str(tip_track['counts'][m20]+tip_track['counts'][p20]))
ctx.comment('20 ul Used racks in total: ' + str((tip_track['counts'][m20]+tip_track['counts'][p20]) / 96))
if (STEPS[2]['Execute'] == True & STEPS[4]['Execute'] == True):
ctx.comment('200 ul Used tips in total: ' + str(tip_track['counts'][p300]))
ctx.comment('200 ul Used racks in total: ' + str(tip_track['counts'][p300] / 96))
ctx.comment('20 ul Used tips in total: ' + str(tip_track['counts'][m20]))
ctx.comment('20 ul Used racks in total: ' + str((tip_track['counts'][m20] / 96)))
if (STEPS[2]['Execute'] == True & STEPS[5]['Execute'] == False):
ctx.comment('200 ul Used tips in total: ' + str(tip_track['counts'][p300]))
ctx.comment('200 ul Used racks in total: ' + str(tip_track['counts'][p300] / 96))
if (STEPS[3]['Execute'] == False & STEPS[4]['Execute'] == True):
ctx.comment('20 ul Used tips in total: ' + str(tip_track['counts'][m20]))
ctx.comment('20 ul Used racks in total: ' + str((tip_track['counts'][m20] / 96)))
ctx.comment('Finished! \nMove plate to PCR')
def run(ctx: protocol_api.ProtocolContext):
ctx.comment('Actual used columns: ' + str(num_cols))
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {
'Execute': True,
'description': 'Transfer Mmix'
},
2: {
'Execute': True,
'description': 'Transfer samples'
},
3: {
'Execute': True,
'description': 'Transfer negative control'
},
4: {
'Execute': True,
'description': 'Transfer positive control'
}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
folder_path = '/var/lib/jupyter/notebooks' + run_id
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/Station_C_Vitro_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self,
name,
flow_rate_aspirate,
flow_rate_dispense,
rinse,
reagent_reservoir_volume,
delay,
num_wells,
h_cono,
v_fondo,
tip_recycling='none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused = []
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
Mmix = Reagent(
name='Mmix',
rinse=False,
flow_rate_aspirate=3,
flow_rate_dispense=3,
reagent_reservoir_volume=1800,
num_wells=1, #change with num samples
delay=0,
h_cono=h_cone,
v_fondo=volume_cone # V cono
)
Samples = Reagent(
name='Samples',
rinse=False,
flow_rate_aspirate=1,
flow_rate_dispense=1,
reagent_reservoir_volume=50,
delay=0,
num_wells=num_cols, # num_cols comes from available columns
h_cono=0,
v_fondo=0)
Mmix.vol_well = Mmix.vol_well_original
Samples.vol_well = Samples.vol_well_original
##################
# Custom functions
def divide_volume(volume, max_vol):
num_transfers = math.ceil(volume / max_vol)
vol_roundup = math.ceil(volume / num_transfers)
last_vol = volume - vol_roundup * (num_transfers - 1)
vol_list = [vol_roundup for v in range(1, num_transfers)]
vol_list.append(last_vol)
return vol_list
def divide_destinations(l, n):
# Divide the list of destinations in size n lists.
for i in range(0, len(l), n):
yield l[i:i + n]
def distribute_custom(pipette,
volume,
src,
dest,
waste_pool,
pickup_height,
extra_dispensal,
dest_x_offset,
disp_height=0):
# Custom distribute function that allows for blow_out in different location and adjustement of touch_tip
pipette.aspirate((len(dest) * volume) + extra_dispensal,
src.bottom(pickup_height))
pipette.touch_tip(speed=20, v_offset=-5)
pipette.move_to(src.top(z=5))
pipette.aspirate(5) # air gap
for d in dest:
pipette.dispense(5, d.top())
drop = d.top(z=disp_height).move(Point(x=dest_x_offset))
pipette.dispense(volume, drop)
pipette.move_to(d.top(z=5))
pipette.aspirate(5) # air gap
try:
pipette.blow_out(waste_pool.wells()[0].bottom(pickup_height + 3))
except:
pipette.blow_out(waste_pool.bottom(pickup_height + 3))
return (len(dest) * volume)
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol,
x_offset, pickup_height, rinse, disp_height,
blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet,
reagent,
location=source,
vol=vol,
rounds=2,
blow_out=True,
mix_height=0,
x_offset=x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x=x_offset[0]))
pipet.aspirate(vol, s) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol,
source.top(z=-2),
rate=reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z=disp_height).move(Point(x=x_offset[1]))
pipet.dispense(vol + air_gap_vol,
drop,
rate=reagent.flow_rate_dispense) # dispense all
ctx.delay(
seconds=reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z=-2))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-5, radius=0.5)
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
x_offset,
source_height=3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height <= 0:
mix_height = 3
pipet.aspirate(1,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
pipet.dispense(vol,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
pipet.dispense(1,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
def calc_height(reagent,
cross_section_area,
aspirate_volume,
min_height=0.5):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
####################################
# load labware and modules
# 24 well rack
tuberack = ctx.load_labware(
'opentrons_24_aluminumblock_generic_2ml_screwcap', '2',
'Opentrons 24 Well Aluminum Block with Generic 2 mL Screwcap')
############################################
# tempdecks
tempdeck_orig = ctx.load_module('Temperature Module Gen2', '4')
tempdeck_dest = ctx.load_module('Temperature Module Gen2', '1')
if SET_TEMP_ON_SLOT_4:
tempdeck_orig.set_temperature(TEMPERATURE_SLOT_4)
if SET_TEMP_ON_SLOT_1:
tempdeck_dest.set_temperature(TEMPERATURE_SLOT_1)
##################################
# Sample plate - comes from B
source_plate = tempdeck_orig.load_labware(
'kingfisher_96_aluminumblock_200ul',
'Kingfisher 96 Aluminum Block 200 uL')
samples = source_plate.wells()[:NUM_SAMPLES]
##################################
# qPCR plate - final plate, goes to PCR
qpcr_plate = tempdeck_dest.load_labware(
'opentrons_96_aluminumblock_nest_wellplate_100ul',
'Opentrons 96 Well Aluminum Block with NEST Well Plate 100 uL')
##################################
# Load Tipracks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['5']
]
tips200 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['3']
]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
Mmix.reagent_reservoir = tuberack.rows()[0][0] # A1
# setup up sample sources and destinations
samples = source_plate.wells()[2:NUM_SAMPLES]
pcr_wells = qpcr_plate.wells()[:NUM_SAMPLES]
pcr_wells_samples = qpcr_plate.wells()[2:NUM_SAMPLES]
# Divide destination wells in small groups for P300 pipette
dests = list(divide_destinations(pcr_wells, size_transfer))
# pipettes
p20 = ctx.load_instrument('p20_single_gen2',
mount='right',
tip_racks=tips20)
p300 = ctx.load_instrument('p300_single_gen2',
mount='left',
tip_racks=tips200)
# used tip counter and set maximum tips available
tip_track = {
'counts': {
p300: 0,
p20: 0
},
'maxes': {
p300: 96 * len(p300.tip_racks),
p20: 96 * len(p20.tip_racks)
}
}
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) +
'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
if not pip.hw_pipette['has_tip']:
pip.pick_up_tip()
##########
############################################################################
# STEP 1: TRANSFER MMIX
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
pick_up(p300)
used_vol = []
for dest in dests:
aspirate_volume = MMIX_VOL_PER_SAMPLE * len(dest) + extra_dispensal
used_vol_temp = distribute_custom(
p300,
volume=MMIX_VOL_PER_SAMPLE,
src=Mmix.reagent_reservoir,
dest=dest,
waste_pool=Mmix.reagent_reservoir,
pickup_height=0.2,
extra_dispensal=extra_dispensal,
dest_x_offset=2,
disp_height=-1)
used_vol.append(used_vol_temp)
p300.drop_tip(home_after=False)
tip_track['counts'][p300] += 1
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: TRANSFER SAMPLES
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
for s, d in zip(samples, pcr_wells_samples):
pick_up(p20)
move_vol_multichannel(p20,
reagent=Samples,
source=s,
dest=d,
vol=VOLUME_SAMPLE,
air_gap_vol=air_gap_sample,
x_offset=x_offset,
pickup_height=0.2,
disp_height=0,
rinse=False,
blow_out=True,
touch_tip=True)
p20.drop_tip(home_after=False)
tip_track['counts'][p20] += 1
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 3: TRANSFER NEGATIVE CONTROL
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
pick_up(p20)
s = tuberack.rows()[0][1] # A2
d = qpcr_plate.wells()[1] # B1
move_vol_multichannel(p20,
reagent=Samples,
source=s,
dest=d,
vol=VOLUME_SAMPLE,
air_gap_vol=air_gap_sample,
x_offset=x_offset,
pickup_height=0.2,
disp_height=0,
rinse=False,
blow_out=True,
touch_tip=True)
p20.drop_tip(home_after=False)
tip_track['counts'][p20] += 1
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 4: TRANSFER POSITIVE CONTROL
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
pick_up(p20)
s = tuberack.rows()[0][2] # A3
d = qpcr_plate.wells()[0] # A1
move_vol_multichannel(p20,
reagent=Samples,
source=s,
dest=d,
vol=VOLUME_SAMPLE,
air_gap_vol=air_gap_sample,
x_offset=x_offset,
pickup_height=0.2,
disp_height=0,
rinse=False,
blow_out=True,
touch_tip=True)
p20.drop_tip(home_after=False)
tip_track['counts'][p20] += 1
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
for i in range(3):
#ctx._hw_manager.hardware.set_lights(rails=False)
ctx._hw_manager.hardware.set_lights(button=(1, 0, 0))
time.sleep(0.3)
#ctx._hw_manager.hardware.set_lights(rails=True)
ctx._hw_manager.hardware.set_lights(button=(0, 0, 1))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=(0, 1, 0))
ctx.comment('Finished! \nMove plate to PCR')
total_used_vol = np.sum(used_vol)
total_needed_volume = total_used_vol
ctx.comment('Total Mmix used volume is: ' + str(total_used_vol) +
'\u03BCl.')
ctx.comment('Needed Mmix volume is ' +
str(total_needed_volume + extra_dispensal * len(dests)) +
'\u03BCl')
ctx.comment('Mmix remaining in tubes is: ' + format(
np.sum(Mmix.unused) + extra_dispensal * len(dests) + Mmix.vol_well) +
'\u03BCl.')
ctx.comment('200 ul Used tips in total: ' + str(tip_track['counts'][p300]))
ctx.comment('200 ul Used racks in total: ' +
str(tip_track['counts'][p300] / 96))
ctx.comment('20 ul Used tips in total: ' + str(tip_track['counts'][p20]))
ctx.comment('20 ul Used racks in total: ' +
str(tip_track['counts'][p20] / 96))
def run(ctx: protocol_api.ProtocolContext):
ctx.comment('Columnas a utilizar: ' + str(num_cols))
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {
'Execute': True,
'description': 'Transferir muestras a la placa PCR'
}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
folder_path = '/var/lib/jupyter/notebooks' + run_id
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/Station_C_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self,
name,
flow_rate_aspirate,
flow_rate_dispense,
rinse,
reagent_reservoir_volume,
delay,
num_wells,
h_cono,
v_fondo,
tip_recycling='none'):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused = []
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
Samples = Reagent(
name='Samples',
rinse=False,
flow_rate_aspirate=sample_aspirate_rate,
flow_rate_dispense=sample_dispense_rate,
reagent_reservoir_volume=50,
delay=0,
num_wells=num_cols, # num_cols comes from available columns
h_cono=0,
v_fondo=0)
Samples.vol_well = Samples.vol_well_original
##################
# Custom functions
def log_parameters():
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('VALORES DE VARIABLES')
ctx.comment(' ')
ctx.comment('Número de muestras: ' + str(NUM_SAMPLES))
ctx.comment('Volumen a transferir a la placa PCR: ' +
str(VOLUME_PCR_SAMPLE) + ' ul')
ctx.comment('Foto-sensible: ' + str(PHOTOSENSITIVE))
ctx.comment('###############################################')
ctx.comment(' ')
def start_run():
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Empezando protocolo')
if PHOTOSENSITIVE == False:
ctx._hw_manager.hardware.set_lights(button=True, rails=True)
else:
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
now = datetime.now()
# dd/mm/YY H:M:S
start_time = now.strftime("%Y/%m/%d %H:%M:%S")
return start_time
def finish_run():
ctx.comment('###############################################')
ctx.comment('Protocolo finalizado')
ctx.comment(' ')
#Set light color to blue
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
now = datetime.now()
# dd/mm/YY H:M:S
finish_time = now.strftime("%Y/%m/%d %H:%M:%S")
if PHOTOSENSITIVE == False:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button=False, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=True)
time.sleep(0.3)
else:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button=False, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
ctx.comment('Puntas de 20 ul utilizadas: ' +
str(tip_track['counts'][p20]) + ' (' +
str(round(tip_track['counts'][p20] / 96, 2)) + ' caja(s))')
ctx.comment('###############################################')
return finish_time
def log_step_start():
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('PASO ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
return datetime.now()
def log_step_end(start):
end = datetime.now()
time_taken = (end - start)
STEPS[STEP]['Time:'] = str(time_taken)
ctx.comment(' ')
ctx.comment('Paso ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' hizo un tiempo de ' + str(time_taken))
ctx.comment(' ')
def move_vol_multichannel(pipet,
reagent,
source,
dest,
vol,
air_gap_vol,
x_offset,
pickup_height,
rinse,
disp_height,
blow_out,
touch_tip,
v_offset=-5,
radius=0.5):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet,
reagent,
location=source,
vol=vol,
rounds=2,
blow_out=True,
mix_height=0,
x_offset=x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x=x_offset[0]))
pipet.aspirate(vol, s) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol,
source.top(z=-2),
rate=reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z=disp_height).move(Point(x=x_offset[1]))
pipet.dispense(vol + air_gap_vol,
drop,
rate=reagent.flow_rate_dispense) # dispense all
ctx.delay(
seconds=reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z=-2))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=v_offset, radius=radius)
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
x_offset,
source_height=3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height <= 0:
mix_height = 3
pipet.aspirate(1,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=source_height).move(
Point(x=x_offset[0])),
rate=reagent.flow_rate_aspirate)
pipet.dispense(vol,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
pipet.dispense(1,
location=location.bottom(z=mix_height).move(
Point(x=x_offset[1])),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
####################################
# load labware and modules
##################################
# Sample plate - comes from B
#source_plate1 = ctx.load_labware(
# 'nest_96_wellplate_100ul_pcr_full_skirt', '1',
# 'NEST 96 Well Plate 100 uL PCR Full Skirt')
source_plate1 = ctx.load_labware(
'opentrons_96_aluminumblock_generic_pcr_strip_200ul', '1',
'Opentrons 96 Well Aluminum Block with Generic PCR Strip 200 µL')
source_plate2 = ctx.load_labware(
'opentrons_96_aluminumblock_generic_pcr_strip_200ul', '2',
'Opentrons 96 Well Aluminum Block with Generic PCR Strip 200 µL')
samples_source1 = source_plate1.columns()[
0::2] # Select odd columns from source plate
samples_source2 = source_plate2.columns()[
0::2] # Select odd columns from source plate
samples_source1 = [
well for columns in samples_source1 for well in columns
] # list of lists to list
samples_source2 = [well for columns in samples_source2 for well in columns]
samples = samples_source1 + samples_source2
samples = samples[:NUM_SAMPLES]
##################################
# qPCR plate - final plate, goes to PCR
qpcr_plate = ctx.load_labware(
'opentrons_96_aluminumblock_generic_pcr_strip_200ul', '4',
'Opentrons 96 Well Aluminum Block with Generic PCR Strip 200 µL')
##################################
# Load Tipracks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['7']
]
################################################################################
# setup up sample sources and destinations
pcr_wells = qpcr_plate.wells()[pcr_plate_well_offset:NUM_SAMPLES +
pcr_plate_well_offset]
# pipettes
p20 = ctx.load_instrument('p20_single_gen2',
mount='right',
tip_racks=tips20)
# used tip counter and set maximum tips available
tip_track = {'counts': {p20: 0}, 'maxes': {p20: 96 * len(p20.tip_racks)}}
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Reemplaza las cajas de puntas de ' +
str(pip.max_volume) + 'µl antes \
de continuar.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
if not pip.hw_pipette['has_tip']:
pip.pick_up_tip()
##########
log_parameters()
start_run()
############################################################################
# STEP 1: TRANSFER SAMPLES
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = log_step_start()
for source, dest in zip(samples, pcr_wells):
pick_up(p20)
move_vol_multichannel(p20,
reagent=Samples,
source=source,
dest=dest,
vol=VOLUME_PCR_SAMPLE + 5,
air_gap_vol=air_gap_pcr_sample,
x_offset=x_offset,
pickup_height=0.1,
disp_height=pcr_disp_height,
v_offset=pcr_disp_height,
rinse=False,
blow_out=True,
touch_tip=dispense_touch_tip,
radius=1)
if dispense_touch_tip == False:
p20.aspirate(air_gap_vol)
if recycle_tip:
p20.return_tip()
else:
p20.drop_tip(home_after=False)
tip_track['counts'][p20] += 1
log_step_end(start)
############################################################################
finish_run()
def run(ctx: protocol_api.ProtocolContext):
import os
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {'Execute': True, 'description': 'Add samples ('+str(volume_sample)+'ul)'},
2: {'Execute': False, 'description': 'Add internal control one by one (10ul)'}
# We won't use it as we will do it in KB with the multichannel pipette
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
if not ctx.is_simulating():
# Folder and file_path for log time
folder_path = '/var/lib/jupyter/notebooks/'+run_id
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/KA_SampleSetup_viral_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
reagent_reservoir_volume, delay, num_wells, h_cono, v_fondo,
tip_recycling = 'none',rinse_loops = 2):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused=[]
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
self.rinse_loops = rinse_loops
Samples = Reagent(name = 'Samples',
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
rinse = False,
delay = 0,
reagent_reservoir_volume = 100 * 24,
num_wells = 24, # num_cols comes from available columns
h_cono = h_cone,
v_fondo = volume_cone
) # cone
IC = Reagent(name = 'Internal control',
flow_rate_aspirate = 1,
flow_rate_dispense = 3,
rinse = False,
delay = 0,
reagent_reservoir_volume = $IC_total_volume,
num_wells = $IC_wells, # num_cols comes from available columns
h_cono = h_cone,
v_fondo = volume_cone
) # cone
Samples.vol_well = Samples.vol_well_original
IC.vol_well = IC.vol_well_original
##################
# Custom functions
def generate_source_table(source):
'''
Concatenate the wells from the different origin racks
'''
for rack_number in range(len(source)):
if rack_number == 0:
s = source[rack_number].wells()
else:
s = s + source[rack_number].wells()
return s
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip,
post_dispense=False, post_dispense_vol=20,
post_airgap=False, post_airgap_vol=10):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = 2, blow_out = True, mix_height = 0,
x_offset = x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s, rate = reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z = -2))
if post_dispense == True:
pipet.dispense(post_dispense_vol, dest.top(z = -2))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -5, radius = 0.9)
if post_airgap == True:
pipet.aspirate(post_airgap_vol, dest.top(z = 5))
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
x_offset, source_height = 3, post_airgap=False, post_airgap_vol=10,
post_dispense=False, post_dispense_vol=20,):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate)
pipet.dispense(vol, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
pipet.dispense(1, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
if post_dispense == True:
pipet.dispense(post_dispense_vol, location.top(z = -2))
if post_airgap == True:
pipet.dispense(post_airgap_vol, location.top(z = 5))
def calc_height(reagent, cross_section_area, aspirate_volume, min_height = 0.4, extra_volume = 50):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume + extra_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
####################################
# load labware and modules
####################################
# Load Sample racks
if NUM_SAMPLES < 96:
rack_num = math.ceil(NUM_SAMPLES / 24)
ctx.comment('Used source racks are ' + str(rack_num))
samples_last_rack = NUM_SAMPLES - rack_num * 24
else:
rack_num = 4
source_tube_types={'Screwcap 2ml': ['opentrons_24_tuberack_generic_2ml_screwcap','source tuberack with screwcap'],
'Eppendorf 1.5ml': ['opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap','source tuberack with eppendorf'],
}
source_racks = [ctx.load_labware(
source_tube_types[source_type][0], slot,
source_tube_types[source_type][1] + str(i + 1)) for i, slot in enumerate(['4', '1', '6', '3'][:rack_num])
]
ic_source_rack = ctx.load_labware('opentrons_24_tuberack_generic_2ml_screwcap', '9',
'Internal control source')
ic_source = ic_source_rack.wells()[0] # internal control comes from 1 bottle
##################################
# Destination plate
dest_plate = ctx.load_labware(
'kf_96_wellplate_2400ul', '5', 'KF 96well destination plate')
####################################
# Load tip_racks
# tips20 = [ctx.load_labware('opentrons_96_filtertiprack_20ul', slot, '20µl filter tiprack')
# for slot in ['2', '8']]
tips300 = [ctx.load_labware('opentrons_96_filtertiprack_200ul', slot, '200µl filter tiprack')
for slot in ['10', '11']]
tips20 = [ctx.load_labware('opentrons_96_filtertiprack_20ul', slot, '20µl filter tiprack')
for slot in ['8']]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
# setup samples and destinations
sample_sources_full = generate_source_table(source_racks)
sample_sources = sample_sources_full[:NUM_SAMPLES]
destinations = dest_plate.wells()[:NUM_SAMPLES]
p20 = ctx.load_instrument(
'p20_single_gen2', mount='left', tip_racks=tips20)
p300 = ctx.load_instrument(
'p300_single_gen2', mount='right', tip_racks=tips300) # load P1000 pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {p300: 0, p20: 0}, # p1000: 0},
'maxes': {p300: len(tips300) * 96, p20: len(tips20)*96} # ,p20: len(tips20)*96,
}
############################################################################
# STEP 1: Add Samples
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
# Transfer parameters
start = datetime.now()
for s, d in zip(sample_sources, destinations):
if not p300.hw_pipette['has_tip']:
pick_up(p300)
# Mix the sample BEFORE dispensing
#custom_mix(p1000, reagent = Samples, location = s, vol = volume_sample, rounds = 2, blow_out = True, mix_height = 15)
move_vol_multichannel(p300, reagent = Samples, source = s, dest = d,
vol=volume_sample, air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 0.3, rinse = Samples.rinse, disp_height = -10,
blow_out = True, touch_tip = True)
# Mix the sample AFTER dispensing
#custom_mix(p300, reagent = Samples, location = d, vol = volume_sample, rounds = 2, blow_out = True, mix_height = 15)
# Drop tip and update counter
p300.drop_tip(home_after=False)
tip_track['counts'][p300] += 1
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: Add internal control
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
#BEWARE, everything with the same tip, dispensed from top!
# Transfer parameters
start = datetime.now()
for d in destinations:
if not p20.hw_pipette['has_tip']:
pick_up(p20)
# Mix the sample BEFORE dispensing
#custom_mix(p1000, reagent = Samples, location = s, vol = volume_sample, rounds = 2, blow_out = True, mix_height = 15)
move_vol_multichannel(p20, reagent = IC, source = ic_source, dest = d,
vol = ic_volume, air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 0.4, rinse = IC.rinse, disp_height = -8,
blow_out = True, touch_tip = False)
# Mix the sample AFTER dispensing
#custom_mix(p20, reagent = Samples, location = d, vol = 10, rounds = 2,
#blow_out = True, mix_height = 2, x_offset = x_offset)
# Drop tip and update counter
p20.drop_tip()
tip_track['counts'][p20] += 1
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
'''if not ctx.is_simulating():
os.system('mpg123 -f -8000 /etc/audio/speaker-test.mp3 &')'''
for i in range(3):
ctx._hw_manager.hardware.set_lights(rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails=True)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails=False)
ctx.home()
ctx.comment(
'Finished! \nMove deepwell plate to Station B.')
ctx.comment('Used 200µl tips in total: ' + str(tip_track['counts'][p300]))
ctx.comment('Used 200ul racks in total: '+str(tip_track['counts'][p300] / 96))
ctx.comment('Used p20 tips in total: ' + str(tip_track['counts'][p20]))
ctx.comment('Used p20 racks in total: ' + str(tip_track['counts'][p20] / 96))
def run(ctx: protocol_api.ProtocolContext):
ctx.comment('Actual used columns: ' + str(num_cols))
#from opentrons.drivers.rpi_drivers import gpio
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {'Execute': True, 'description': 'Add 50 ul Elution Buffer'},
2: {'Execute': True, 'description': 'Add 100 ul Wash Buffer 1 - Round 1'},
3: {'Execute': True, 'description': 'Add 100 ul Wash Buffer 2 - Round 1 and stop until plate comes from A'},
4: {'Execute': True, 'description': 'Add 100 ul Lysis Buffer and then move to station A'},
5: {'Execute': False, 'description': 'Transfer IC'},
6: {'Execute': True, 'description': 'Transfer ICtwo'},
7: {'Execute': False, 'description': 'Mix beads'},
8: {'Execute': False, 'description': 'Transfer beads'},
9: {'Execute': True, 'description': 'Mix beadstwo'},
10: {'Execute': True, 'description': 'Transfer beadstwo'}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
folder_path = '/var/lib/jupyter/notebooks/'+str(run_id)
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/KB_station_viral_time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
reagent_reservoir_volume, delay, num_wells, h_cono, v_fondo,
tip_recycling = 'none', rinse_loops = 3, flow_rate_dispense_mix = 4, flow_rate_aspirate_mix = 4):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.flow_rate_aspirate_mix = flow_rate_aspirate_mix
self.flow_rate_dispense_mix = flow_rate_dispense_mix
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay #Delay of reagent in dispense
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.unused=[]
self.tip_recycling = tip_recycling
self.vol_well_original = reagent_reservoir_volume / num_wells
self.rinse_loops = rinse_loops
# Reagents and their characteristics
WashBuffer1 = Reagent(name='Wash Buffer 1',
flow_rate_aspirate=0.75,
flow_rate_dispense=1,
rinse=True,
rinse_loops=6,
delay=3,
reagent_reservoir_volume=$Wone_total_volume, #100*NUM_SAMPLES,
num_wells=$Wone_wells,
h_cono=1.95,
v_fondo=695) # Flat surface
WashBuffer2 = Reagent(name='Wash Buffer 2',
flow_rate_aspirate=0.75,
flow_rate_dispense=1,
rinse=True,
delay=3,
reagent_reservoir_volume=$Wtwo_total_volume, #100*NUM_SAMPLES,
num_wells=$Wtwo_wells,
h_cono=1.95,
v_fondo=695) # Flat surface
Lysis = Reagent(name='Lysis Buffer',
flow_rate_aspirate=0.75,
flow_rate_dispense=0.5,
rinse=False,
rinse_loops=6,
delay=2,
reagent_reservoir_volume=$Lysis_total_volume, #100*NUM_SAMPLES,
num_wells=1,
h_cono=1.95,
v_fondo=695) # Flat surface
ElutionBuffer = Reagent(name='Elution Buffer',
flow_rate_aspirate=1,
flow_rate_dispense=1,
rinse=False,
delay=0,
reagent_reservoir_volume=$Elution_total_volume,#50*NUM_SAMPLES,
num_wells=$Elution_wells,
h_cono=1.95,
v_fondo=695) # Prismatic
IC = Reagent(name='Magnetic beads and Lysis',
flow_rate_aspirate=1,
flow_rate_dispense=3,
rinse=False,
num_wells=$IC_wells,
delay=2,
reagent_reservoir_volume=$IC_total_volume,#20 * NUM_SAMPLES * 1.1,
h_cono=1.95,
v_fondo=695) # Prismatic
ICtwo = Reagent(name='Internal control 2',
flow_rate_aspirate=1,
flow_rate_dispense=3,
rinse=False,
num_wells=1,
delay=2,
reagent_reservoir_volume=$IC_total_volume,#20 * NUM_SAMPLES * 1.1,
h_cono=1,
v_fondo=10) # Prismatic
Beads = Reagent(name='Magnetic beads and Lysis',
flow_rate_aspirate=0.5,
flow_rate_dispense=0.5,
rinse=True,
rinse_loops=6,
num_wells=$Beads_wells,
delay=3,
reagent_reservoir_volume=$Beads_total_volume,#20 * NUM_SAMPLES * 1.1,
h_cono=1.95,
v_fondo=695) # Prismatic
Beadstwo = Reagent(name='Magnetic beads 2',
flow_rate_aspirate=0.5,
flow_rate_dispense=0.5,
rinse=True,
rinse_loops=4,
num_wells=2,
delay=3,
reagent_reservoir_volume=$Beads_total_volume,#20 * NUM_SAMPLES * 1.1,
h_cono=1.95,
v_fondo=695) # Prismatic
Beads.vol_well = Beads.vol_well_original
IC.vol_well = IC.vol_well_original
WashBuffer1.vol_well = WashBuffer1.vol_well_original
WashBuffer2.vol_well = WashBuffer2.vol_well_original
ElutionBuffer.vol_well = ElutionBuffer.vol_well_original
Lysis.vol_well = Lysis.vol_well_original
Beadstwo.vol_well = Beadstwo.vol_well_original
ICtwo.vol_well = ICtwo.vol_well_original
##################
# Custom functions
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip,
post_dispense=False, post_dispense_vol=20,
post_airgap=True, post_airgap_vol=10):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = reagent.rinse_loops, blow_out = True, mix_height = 0,
x_offset = x_offset, post_airgap=False,post_dispense=False, post_dispense_vol=20,post_airgap_vol=10)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s, rate = reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z = -5))
if post_airgap == True:
pipet.aspirate(post_airgap_vol, dest.top(z = -2))
if post_dispense == True:
pipet.dispense(post_dispense_vol, dest.top(z = -2))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -5, radius = 0.9)
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
source_height = 3, post_airgap=True, post_airgap_vol=10,
post_dispense=False, post_dispense_vol=20,x_offset = x_offset):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height == 0:
mix_height = 3
pipet.aspirate(1, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate_mix)
for _ in range(rounds):
pipet.aspirate(vol, location=location.bottom(
z=source_height).move(Point(x=x_offset[0])), rate=reagent.flow_rate_aspirate_mix)
pipet.dispense(vol, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense_mix)
pipet.dispense(1, location=location.bottom(
z=mix_height).move(Point(x=x_offset[1])), rate=reagent.flow_rate_dispense_mix)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
if post_dispense == True:
pipet.dispense(post_dispense_vol, location.top(z = -2))
if post_airgap == True:
pipet.aspirate(post_airgap_vol, location.top(z = 5))
def calc_height(reagent, cross_section_area, aspirate_volume, min_height = 0.2, extra_volume = 50):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if reagent.vol_well < aspirate_volume + extra_volume:
reagent.unused.append(reagent.vol_well)
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume - reagent.v_cono) / cross_section_area #- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
##########
def find_side(col):
'''
Detects if the current column has the magnet at its left or right side
'''
if col % 2 == 0:
side = -1 # left
else:
side = 1
return side
####################################
# load labware and modules
# IC well rack
####################################
#tempdeck = ctx.load_module('tempdeck', '3')
ic_reservoir = ctx.load_labware(
'nest_96_wellplate_100ul_pcr_full_skirt','3', 'Wellplate with Beads and IC')
# 12 well rack
####################################
reagent_res = ctx.load_labware(
'nest_12_reservoir_15ml', '5', 'Reservoir 12 channel, column 1')
# Wash Buffer 1 100ul Deepwell plate
############################################
WashBuffer1_100ul_plate1 = ctx.load_labware(
'kf_96_wellplate_2400ul', '1', 'Wash Buffer 1 Deepwell plate 1')
# Wash Buffer 2 100ul Deepwell plate
############################################
WashBuffer2_100ul_plate1 = ctx.load_labware(
'kf_96_wellplate_2400ul', '4', 'Wash Buffer 2 Deepwell plate 1')
# Lysis buffer 100ul Deepwell plate and later will be the plate with samples
############################################
kf_plate = ctx.load_labware(
'kf_96_wellplate_2400ul', '2', 'Sample Deepwell plate 1')
# Elution Deepwell plate
############################################
ElutionBuffer_50ul_plate = ctx.load_labware(
'kingfisher_std_96_wellplate_550ul', '7', 'Elution Buffer 50 ul STD plate')
####################################
# Load tip_racks
tips300 = [ctx.load_labware('opentrons_96_tiprack_300ul', slot, '200µl filter tiprack')
for slot in ['8']]
tips20 = [ctx.load_labware('opentrons_96_filtertiprack_20ul', slot, '20µl filter tiprack')
for slot in ['6','9']]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
WashBuffer1.reagent_reservoir = reagent_res.rows(
)[0][:1] # position 1
WashBuffer2.reagent_reservoir = reagent_res.rows(
)[0][1:2] #position 2
ElutionBuffer.reagent_reservoir = reagent_res.rows(
)[0][9:10] #position 10
Lysis.reagent_reservoir = reagent_res.rows(
)[0][2:3] #position 3
IC.reagent_reservoir = reagent_res.rows(
)[0][10:11] #position 4
Beads.reagent_reservoir = reagent_res.rows(
)[0][11:] #position 4
ICtwo.reagent_reservoir = ic_reservoir.rows(
)[0][:1] #position 1
Beadstwo.reagent_reservoir = ic_reservoir.rows(
)[0][1:3] #position 2 to 3
# columns in destination plates to be filled depending the number of samples
wb1plate1_destination = WashBuffer1_100ul_plate1.rows()[0][:num_cols]
wb2plate1_destination = WashBuffer2_100ul_plate1.rows()[0][:num_cols]
elutionbuffer_destination = ElutionBuffer_50ul_plate.rows()[0][:num_cols]
kf_destination = kf_plate.rows()[0][:num_cols]
# pipette
m300 = ctx.load_instrument(
'p300_multi_gen2', 'right', tip_racks=tips300) # Load multi pipette
# pipettes. P1000 currently deactivated
m20 = ctx.load_instrument(
'p20_multi_gen2', 'left', tip_racks=tips20) # Load p300 multi pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {m300: 0, m20: 0},
'maxes': {m300: len(tips300)*96, m20: len(tips20)*96}
}
############################################################################
# STEP 1: Transfer Elution buffer
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
# Elution buffer
ElutionBuffer_vol = [Elution_vol]
########
# Water or elution buffer
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for transfer_vol in ElutionBuffer_vol:
# Calculate pickup_height based on remaining volume and shape of container
move_vol_multichannel(m300, reagent = ElutionBuffer, source = ElutionBuffer.reagent_reservoir[ElutionBuffer.col],
dest = elutionbuffer_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol_elutionbuffer, x_offset = x_offset,
pickup_height = 0.2, rinse = False, disp_height = -2,
blow_out = True, touch_tip = True, post_airgap=True)
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: Filling with WashBuffer1
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
WB1 = [WBone_vol]
rinse = False # Only first time
########
# Wash buffer dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(WB1):
if (i == 0 and j == 0):
rinse = True #Rinse only first transfer
else:
rinse = False
move_vol_multichannel(m300, reagent = WashBuffer1, source = WashBuffer1.reagent_reservoir[WashBuffer1.col],
dest = wb1plate1_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 0.3, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = False, post_airgap=True)
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 3: Filling with WashBuffer2
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
WB2 = [WBtwo_vol]
rinse = False # Only first time
########
# Wash buffer dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(WB2):
if (i == 0 and j == 0):
rinse = True
else:
rinse = False
move_vol_multichannel(m300, reagent = WashBuffer2, source = WashBuffer2.reagent_reservoir[WashBuffer2.col],
dest = wb2plate1_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 0.2, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = False, post_airgap=True)
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
ctx.pause('Bring plate from A in position 2 and click continue. Put IC and Beads in position 3')
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 4: Filling with Lysis
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
lysis_vol = [100]
rinse = False # Only first time
########
# Wash buffer dispense
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(lysis_vol):
if (i == 0 and j == 0):
rinse = True #Rinse only first transfer
else:
rinse = False
move_vol_multichannel(m300, reagent = Lysis, source = Lysis.reagent_reservoir[Lysis.col],
dest = kf_destination[i], vol = transfer_vol,
air_gap_vol = air_gap_vol, x_offset = x_offset,
pickup_height = 0.2, rinse = rinse, disp_height = -2,
blow_out = True, touch_tip = False, post_airgap=True,post_dispense=True)
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 5: TRANSFER IC
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
# Transfer parameters
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
IC_transfer_vol = [ic_vol]
rinse = True
for i in range(num_cols):
if not m20.hw_pipette['has_tip']:
pick_up(m20)
for j, transfer_vol in enumerate(IC_transfer_vol):
move_vol_multichannel(m20, reagent=IC, source=IC.reagent_reservoir[IC.col],
dest=kf_destination[i], vol=transfer_vol,
air_gap_vol=air_gap_ic, x_offset=[0,0],
pickup_height=0.2, disp_height = -40.7,
rinse=IC.rinse, blow_out = True, touch_tip=False, post_airgap=True)
m20.drop_tip(home_after=False)
tip_track['counts'][m20] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 6: TRANSFER IC2
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
# Transfer parameters
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
IC_transfer_vol = [ic_vol]
rinse = True
for i in range(num_cols):
if not m20.hw_pipette['has_tip']:
pick_up(m20)
for j, transfer_vol in enumerate(IC_transfer_vol):
move_vol_multichannel(m20, reagent=ICtwo, source=ICtwo.reagent_reservoir[IC.col],
dest=kf_destination[i], vol=transfer_vol,
air_gap_vol=air_gap_ic, x_offset=[0,0],
pickup_height=0.5, disp_height = -40.7,
rinse=ICtwo.rinse, blow_out = True, touch_tip=False, post_airgap=True)
m20.drop_tip(home_after=False)
tip_track['counts'][m20] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 7: PREMIX BEADS
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
if not m300.hw_pipette['has_tip']:
pick_up(m300)
ctx.comment('Tip picked up')
ctx.comment('Mixing ' + Beads.name)
# Mixing
custom_mix(m300, Beads, Beads.reagent_reservoir[Beads.col], vol=120,
rounds=10, blow_out=True, mix_height=10, post_dispense=True, source_height=0.3)
ctx.comment('Finished premixing!')
ctx.comment('Now, reagents will be transferred to deepwell plate.')
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 8: TRANSFER BEADS
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
# Transfer parameters
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
beads_transfer_vol = [beads_vol] # Two rounds of 130
rinse = True
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(beads_transfer_vol):
if (i == 0 and j == 0):
rinse = True
else:
rinse = False
# Calculate pickup_height based on remaining volume and shape of container
[pickup_height, change_col] = calc_height(
reagent = Beads, cross_section_area = multi_well_rack_area,
aspirate_volume = transfer_vol * 8, min_height=0.3, extra_volume=10)
if change_col == True: # If we switch column because there is not enough volume left in current reservoir column we mix new column
ctx.comment(
'Mixing new reservoir column: ' + str(Beads.col))
custom_mix(m300, Beads, Beads.reagent_reservoir[Beads.col],
vol=120, rounds=10, blow_out=True, mix_height=1,
post_dispense=True)
ctx.comment(
'Aspirate from reservoir column: ' + str(Beads.col))
ctx.comment('Pickup height is ' + str(pickup_height))
move_vol_multichannel(m300, reagent=Beads, source=Beads.reagent_reservoir[Beads.col],
dest=kf_destination[i], vol=transfer_vol,
air_gap_vol=air_gap_vol, x_offset=x_offset,
pickup_height=0.2, disp_height = -8,
rinse=rinse, blow_out = True, touch_tip=False, post_airgap=True)
'''custom_mix(m300, Beads, work_destinations_cols[i] ,
vol=70, rounds=10, blow_out=True, mix_height=8,
x_offset = x_offset, source_height=0.5, post_dispense=True)
m300.drop_tip(home_after=False)
'''
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 9: PREMIX BEADStwo
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
if not m300.hw_pipette['has_tip']:
pick_up(m300)
ctx.comment('Tip picked up')
ctx.comment('Mixing ' + Beadstwo.name)
# Mixing
custom_mix(m300, Beadstwo, Beadstwo.reagent_reservoir[Beadstwo.col], vol=80,
rounds=10, blow_out=True, mix_height=5, post_dispense=True, source_height=0.7)
ctx.comment('Finished premixing!')
ctx.comment('Now, reagents will be transferred to deepwell plate.')
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 10: TRANSFER BEADStwo
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
# Transfer parameters
start = datetime.now()
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
beads_transfer_vol = [beads_vol] # Two rounds of 130
rinse = True
for i in range(num_cols):
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(beads_transfer_vol):
if (i == 0 and j == 0):
rinse = True
else:
rinse = False
# Calculate pickup_height based on remaining volume and shape of container
[pickup_height, change_col] = calc_height(
reagent = Beadstwo, cross_section_area = 15,
aspirate_volume = transfer_vol , min_height=0.7, extra_volume=0) # I will consider only aspiration from one well
pickup_height=0.7
if change_col == True: # If we switch column because there is not enough volume left in current reservoir column we mix new column
ctx.comment(
'Mixing new reservoir column: ' + str(Beadstwo.col))
custom_mix(m300, Beadstwo, Beadstwo.reagent_reservoir[Beadstwo.col],
vol=100, rounds=10, blow_out=True, mix_height=0.7,
post_dispense=True)
ctx.comment(
'Aspirate from reservoir column: ' + str(Beadstwo.col))
ctx.comment('Pickup height is ' + str(pickup_height))
move_vol_multichannel(m300, reagent=Beadstwo, source=Beadstwo.reagent_reservoir[Beadstwo.col],
dest=kf_destination[i], vol=transfer_vol,
air_gap_vol=air_gap_vol, x_offset=x_offset,
pickup_height=pickup_height, disp_height = -8,
rinse=rinse, blow_out = True, touch_tip=False, post_airgap=True)
'''custom_mix(m300, Beads, work_destinations_cols[i] ,
vol=70, rounds=10, blow_out=True, mix_height=8,
x_offset = x_offset, source_height=0.5, post_dispense=True)
m300.drop_tip(home_after=False)
'''
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
# Light flash end of program
if not ctx.is_simulating():
for i in range(3):
ctx._hw_manager.hardware.set_lights(rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails=True)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails=False)
ctx.home()
ctx.comment(
'Finished! \nMove deepwell plates to KingFisher extractor.')
ctx.comment('Used tips in total: ' + str(tip_track['counts'][m300]))
ctx.comment('Used racks in total: ' + str(tip_track['counts'][m300] / 96))
def run(ctx: protocol_api.ProtocolContext):
# load labware
dest_plate = ctx.load_labware('nest_96_wellplate_2ml_deep', '2',
'96-deepwell sample plate')
tipracks1000 = [
ctx.load_labware('opentrons_96_filtertiprack_1000ul', '1',
'1000µl filter tiprack')
]
# load pipette
p1000 = ctx.load_instrument('p1000_single_gen2',
'right',
tip_racks=tipracks1000)
tip_log = {'count': {}}
folder_path = '/data/A'
tip_file_path = folder_path + '/tip_log.json'
if TIP_TRACK and not ctx.is_simulating():
if os.path.isfile(tip_file_path):
with open(tip_file_path) as json_file:
data = json.load(json_file)
if 'tips1000' in data:
tip_log['count'][p1000] = data['tips1000']
else:
tip_log['count'][p1000] = 0
else:
tip_log['count'] = {p1000: 0}
tip_log['tips'] = {
p1000: [tip for rack in tipracks1000 for tip in rack.wells()]
}
tip_log['max'] = {pip: len(tip_log['tips'][pip]) for pip in [p1000]}
def pick_up(pip):
nonlocal tip_log
if tip_log['count'][pip] == tip_log['max'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_log['count'][pip] = 0
pip.pick_up_tip(tip_log['tips'][pip][tip_log['count'][pip]])
tip_log['count'][pip] += 1
# pool samples
for i in range(math.ceil(NUM_SAMPLES / 2)):
if NUM_SAMPLES % 2 != 0 and i == math.ceil(NUM_SAMPLES / 2) - 1:
pool_source_set = [dest_plate.wells()[NUM_SAMPLES]]
vol = SAMPLE_VOLUME * 2
else:
pool_source_set = dest_plate.wells()[i * 2:i * 2 + 2]
vol = SAMPLE_VOLUME
for s in pool_source_set:
pick_up(p1000)
p1000.transfer(vol,
s,
dest_plate.wells()[i + 64],
air_gap=20,
new_tip='never')
p1000.air_gap(100)
p1000.drop_tip()
ctx.comment('Move deepwell plate (slot 2) to Station B for RNA \
extraction.')
# track final used tip
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
data = {'tips1000': tip_log['count'][p1000]}
with open(tip_file_path, 'w') as outfile:
json.dump(data, outfile)
def run(ctx: protocol_api.ProtocolContext):
#Change light to red
ctx._hw_manager.hardware.set_lights(button=(1, 0, 0))
ctx.comment('Actual used columns: ' + str(num_cols))
STEP = 0
STEPS = { #Dictionary with STEP activation, description, and times
1: {
'Execute': True,
'description': 'Transfer beads + PK + binding'
},
2: {
'Execute': True,
'description': 'Transfer wash'
},
3: {
'Execute': True,
'description': 'Transfer ethanol'
},
4: {
'Execute': True,
'description': 'Transfer elution'
}
}
#Folder and file_path for log time
import os
folder_path = '/var/lib/jupyter/notebooks' + run_id
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/Station_B_Preparacion_Kingfisher_time_log.txt'
#Define Reagents as objects with their properties
class Reagent:
def __init__(self,
name,
flow_rate_aspirate,
flow_rate_dispense,
flow_rate_aspirate_mix,
flow_rate_dispense_mix,
air_gap_vol_bottom,
air_gap_vol_top,
disposal_volume,
rinse,
max_volume_allowed,
reagent_volume,
reagent_reservoir_volume,
num_wells,
h_cono,
v_fondo,
tip_recycling='none',
dead_vol=700):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.flow_rate_aspirate_mix = flow_rate_aspirate_mix
self.flow_rate_dispense_mix = flow_rate_dispense_mix
self.air_gap_vol_bottom = air_gap_vol_bottom
self.air_gap_vol_top = air_gap_vol_top
self.disposal_volume = disposal_volume
self.rinse = bool(rinse)
self.max_volume_allowed = max_volume_allowed
self.reagent_volume = reagent_volume
self.reagent_reservoir_volume = reagent_reservoir_volume
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.tip_recycling = tip_recycling
self.dead_vol = dead_vol
self.vol_well_original = (reagent_reservoir_volume / num_wells
) + dead_vol if num_wells > 0 else 0
#Reagents and their characteristics
Wash = Reagent(
name='Wash',
flow_rate_aspirate=5, # Original = 0.5
flow_rate_dispense=5, # Original = 1
flow_rate_aspirate_mix=
1, # Liquid density very high, needs slow aspiration
flow_rate_dispense_mix=
1, # Liquid density very high, needs slow dispensation
air_gap_vol_bottom=5,
air_gap_vol_top=0,
disposal_volume=1,
rinse=True,
max_volume_allowed=180,
reagent_volume=
WASH_VOLUME_PER_SAMPLE, # reagent volume needed per sample
reagent_reservoir_volume=(NUM_SAMPLES + 5) *
WASH_VOLUME_PER_SAMPLE, #70000, #51648
num_wells=1,
h_cono=1.95,
v_fondo=695) #1.95 * multi_well_rack_area / 2, #Prismatic
Ethanol = Reagent(
name='Ethanol',
flow_rate_aspirate=5,
flow_rate_dispense=5,
flow_rate_aspirate_mix=1,
flow_rate_dispense_mix=1,
air_gap_vol_bottom=5,
air_gap_vol_top=0,
disposal_volume=1,
rinse=True,
max_volume_allowed=180,
reagent_volume=ETHANOL_VOLUME_PER_SAMPLE,
reagent_reservoir_volume=(NUM_SAMPLES + 5) * ETHANOL_VOLUME_PER_SAMPLE,
num_wells=1,
h_cono=1.95,
v_fondo=695) #1.95 * multi_well_rack_area / 2, #Prismatic
Beads_PK_Binding = Reagent(
name='Magnetic beads + PK + Binding',
flow_rate_aspirate=0.5,
flow_rate_dispense=0.5,
flow_rate_aspirate_mix=0.5,
flow_rate_dispense_mix=0.5,
air_gap_vol_bottom=5,
air_gap_vol_top=0,
disposal_volume=1,
rinse=True,
max_volume_allowed=180,
reagent_volume=BEADS_VOLUME_PER_SAMPLE,
reagent_reservoir_volume=NUM_SAMPLES * BEADS_VOLUME_PER_SAMPLE * 1.1,
num_wells=math.ceil(NUM_SAMPLES * BEADS_VOLUME_PER_SAMPLE * 1.1 /
11500),
h_cono=1.95,
v_fondo=695) #1.95 * multi_well_rack_area / 2, #Prismatic
Elution = Reagent(
name='Elution',
flow_rate_aspirate=3, # Original 0.5
flow_rate_dispense=3, # Original 1
flow_rate_aspirate_mix=15,
flow_rate_dispense_mix=25,
air_gap_vol_bottom=5,
air_gap_vol_top=0,
disposal_volume=1,
rinse=False,
max_volume_allowed=180,
reagent_volume=ELUTION_VOLUME_PER_SAMPLE,
reagent_reservoir_volume=(NUM_SAMPLES + 5) * ELUTION_VOLUME_PER_SAMPLE,
num_wells=math.ceil(
(NUM_SAMPLES + 5) * ELUTION_VOLUME_PER_SAMPLE / 13000),
h_cono=1.95,
v_fondo=695) #1.95 * multi_well_rack_area / 2, #Prismatic
Sample = Reagent(
name='Sample',
flow_rate_aspirate=3, # Original 0.5
flow_rate_dispense=3, # Original 1
flow_rate_aspirate_mix=15,
flow_rate_dispense_mix=25,
air_gap_vol_bottom=5,
air_gap_vol_top=0,
disposal_volume=1,
rinse=False,
max_volume_allowed=150,
reagent_volume=50,
reagent_reservoir_volume=(NUM_SAMPLES + 5) * 50, #14800,
num_wells=num_cols, #num_cols comes from available columns
h_cono=4,
v_fondo=4 * math.pi * 4**3 / 3) #Sphere
Wash.vol_well = Wash.vol_well_original
Ethanol.vol_well = Ethanol.vol_well_original
Beads_PK_Binding.vol_well = Beads_PK_Binding.vol_well_original
Elution.vol_well = Elution.vol_well_original
Sample.vol_well = 350 # Arbitrary value
def str_rounded(num):
return str(int(num + 0.5))
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('VOLUMES FOR ' + str(NUM_SAMPLES) + ' SAMPLES')
ctx.comment(' ')
ctx.comment('Beads + PK + Binding: ' + str(Beads_PK_Binding.num_wells) +
' wells from well 2 in multi reservoir with volume ' +
str_rounded(Beads_PK_Binding.vol_well_original) +
' uL each one')
ctx.comment('Elution: ' + str(Elution.num_wells) +
' wells from well 7 in multi reservoir with volume ' +
str_rounded(Elution.vol_well_original) + ' uL each one')
ctx.comment('Wash: in reservoir 1 with volume ' +
str_rounded(Wash.vol_well_original) + ' uL')
ctx.comment('Etanol: in reservoir 2 with volume ' +
str_rounded(Ethanol.vol_well_original) + ' uL')
ctx.comment('###############################################')
ctx.comment(' ')
###################
#Custom functions
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
offset,
wait_time=0,
drop_height=-1,
two_thirds_mix_bottom=False):
'''
Function for mix in the same location a certain number of rounds. Blow out optional. Offset
can set to 0 or a higher/lower value which indicates the lateral movement
'''
if mix_height <= 0:
mix_height = 1
pipet.aspirate(1,
location=location.bottom(z=mix_height),
rate=reagent.flow_rate_aspirate_mix)
for i in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=mix_height),
rate=reagent.flow_rate_aspirate_mix)
if two_thirds_mix_bottom and i < ((rounds / 3) * 2):
pipet.dispense(vol,
location=location.bottom(z=5).move(
Point(x=offset)),
rate=reagent.flow_rate_dispense_mix)
else:
pipet.dispense(vol,
location=location.top(z=drop_height).move(
Point(x=offset)),
rate=reagent.flow_rate_dispense_mix)
pipet.dispense(1,
location=location.bottom(z=mix_height),
rate=reagent.flow_rate_dispense_mix)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
if wait_time != 0:
ctx.delay(seconds=wait_time,
msg='Waiting for ' + str(wait_time) + ' seconds.')
def calc_height(reagent,
cross_section_area,
aspirate_volume,
min_height=0.4):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if (reagent.vol_well - reagent.dead_vol) < aspirate_volume:
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
def move_vol_multi(pipet,
reagent,
source,
dest,
vol,
x_offset_source,
x_offset_dest,
pickup_height,
rinse,
avoid_droplet,
wait_time,
blow_out,
touch_tip=False,
drop_height=-5):
# Rinse before aspirating
if rinse == True:
#pipet.aspirate(air_gap_vol_top, location = source.top(z = -5), rate = reagent.flow_rate_aspirate) #air gap
custom_mix(pipet,
reagent,
location=source,
vol=vol,
rounds=20,
blow_out=False,
mix_height=3,
offset=0)
#pipet.dispense(air_gap_vol_top, location = source.top(z = -5), rate = reagent.flow_rate_dispense)
# SOURCE
if reagent.air_gap_vol_top != 0: #If there is air_gap_vol, switch pipette to slow speed
pipet.move_to(source.top(z=0))
pipet.air_gap(reagent.air_gap_vol_top) #air gap
#pipet.aspirate(reagent.air_gap_vol_top, source.top(z = -5), rate = reagent.flow_rate_aspirate) #air gap
s = source.bottom(pickup_height).move(Point(x=x_offset_source))
pipet.aspirate(vol, s,
rate=reagent.flow_rate_aspirate) # aspirate liquid
if reagent.air_gap_vol_bottom != 0: #If there is air_gap_vol, switch pipette to slow speed
pipet.move_to(source.top(z=0))
pipet.air_gap(reagent.air_gap_vol_bottom) #air gap
#pipet.aspirate(air_gap_vol_bottom, source.top(z = -5), rate = reagent.flow_rate_aspirate) #air gap
if wait_time != 0:
ctx.delay(seconds=wait_time,
msg='Waiting for ' + str(wait_time) + ' seconds.')
if avoid_droplet == True: # Touch the liquid surface to avoid droplets
ctx.comment("Moving to: " + str(round(pickup_height, 2)) + ' mm')
pipet.move_to(source.bottom(pickup_height))
# GO TO DESTINATION
d = dest.top(z=drop_height).move(Point(x=x_offset_dest))
pipet.dispense(vol - reagent.disposal_volume +
reagent.air_gap_vol_bottom,
d,
rate=reagent.flow_rate_dispense)
if wait_time != 0:
ctx.delay(seconds=wait_time,
msg='Waiting for ' + str(wait_time) + ' seconds.')
if reagent.air_gap_vol_top != 0:
pipet.dispense(reagent.air_gap_vol_top,
dest.top(z=0),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(dest.top(z=-5))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-10, radius=0.7)
#if reagent.air_gap_vol_bottom != 0:
#pipet.move_to(dest.top(z = 0))
#pipet.air_gap(reagent.air_gap_vol_bottom) #air gap
#pipet.aspirate(air_gap_vol_bottom, dest.top(z = 0),rate = reagent.flow_rate_aspirate) #air gap
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
#if not ctx.is_simulating():
if tip_track['counts'][pip] >= tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
##########
def find_side(col):
if col % 2 == 0:
side = -1 # left
else:
side = 1 # right
return side
# load labware and modules
############################################
######## Sample plate - comes from A
deepwell_plate_samples = ctx.load_labware('nest_96_wellplate_2ml_deep',
'1',
'NEST 96 Deepwell Plate 2mL 1')
deepwell_plate_wash = ctx.load_labware('nest_96_wellplate_2ml_deep', '5',
'NEST 96 Deepwell Plate 2mL 2')
deepwell_plate_ethanol = ctx.load_labware('nest_96_wellplate_2ml_deep',
'6',
'NEST 96 Deepwell Plate 2mL 3')
deepwell_plate_elution = ctx.load_labware(
'biorad_96_wellplate_200ul_pcr', '7',
'Bio-Rad 96 Well Plate 200 µL PCR')
####################################
######## 12 well rack
reagent_multi_res = ctx.load_labware('nest_12_reservoir_15ml', '4',
'Reagent deepwell plate')
####################################
######## Single reservoirs
reagent_res_1 = ctx.load_labware('nest_1_reservoir_195ml', '2',
'Single reagent reservoir 1')
res_1 = reagent_res_1.wells()[0]
reagent_res_2 = ctx.load_labware('nest_1_reservoir_195ml', '3',
'Single reagent reservoir 2')
res_2 = reagent_res_2.wells()[0]
####################################
######### Load tip_racks
tips300 = [
ctx.load_labware('opentrons_96_tiprack_300ul', slot,
'200µl filter tiprack') for slot in ['8', '9']
]
###############################################################################
#Declare which reagents are in each reservoir as well as deepwell and sample plate
Wash.reagent_reservoir = res_1
Ethanol.reagent_reservoir = res_2
Beads_PK_Binding.reagent_reservoir = reagent_multi_res.rows()[0][1:5]
Elution.reagent_reservoir = reagent_multi_res.rows()[0][6:7]
work_destinations = deepwell_plate_samples.rows()[0][:Sample.num_wells]
wash_destinations = deepwell_plate_wash.rows()[0][:Sample.num_wells]
ethanol_destinations = deepwell_plate_ethanol.rows()[0][:Sample.num_wells]
elution_destinations = deepwell_plate_elution.rows()[0][:Sample.num_wells]
# pipettes.
m300 = ctx.load_instrument('p300_multi_gen2', 'right',
tip_racks=tips300) # Load multi pipette
#### used tip counter and set maximum tips available
tip_track = {
'counts': {
m300: 0
},
'maxes': {
m300: 96 * len(m300.tip_racks)
} #96 tips per tiprack * number or tipracks in the layout
}
###############################################################################
###############################################################################
###############################################################################
# STEP 1 TRANSFER BEADS + PK + Binding
########
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
beads_trips = math.ceil(Beads_PK_Binding.reagent_volume /
Beads_PK_Binding.max_volume_allowed)
beads_volume = Beads_PK_Binding.reagent_volume / beads_trips #136.66
beads_transfer_vol = []
for i in range(beads_trips):
beads_transfer_vol.append(beads_volume +
Beads_PK_Binding.disposal_volume)
x_offset_source = 0
x_offset_dest = 0
rinse = False # Original: True
first_mix_done = False
for i in range(num_cols):
ctx.comment("Column: " + str(i))
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(beads_transfer_vol):
#Calculate pickup_height based on remaining volume and shape of container
[pickup_height,
change_col] = calc_height(Beads_PK_Binding,
multi_well_rack_area,
transfer_vol * 8)
if change_col == True or not first_mix_done: #If we switch column because there is not enough volume left in current reservoir column we mix new column
ctx.comment('Mixing new reservoir column: ' +
str(Beads_PK_Binding.col))
custom_mix(m300,
Beads_PK_Binding,
Beads_PK_Binding.reagent_reservoir[
Beads_PK_Binding.col],
vol=Beads_PK_Binding.max_volume_allowed,
rounds=BEADS_WELL_FIRST_TIME_NUM_MIXES,
blow_out=False,
mix_height=0.5,
offset=0)
first_mix_done = True
else:
ctx.comment('Mixing reservoir column: ' +
str(Beads_PK_Binding.col))
custom_mix(m300,
Beads_PK_Binding,
Beads_PK_Binding.reagent_reservoir[
Beads_PK_Binding.col],
vol=Beads_PK_Binding.max_volume_allowed,
rounds=BEADS_WELL_NUM_MIXES,
blow_out=False,
mix_height=0.5,
offset=0)
ctx.comment('Aspirate from reservoir column: ' +
str(Beads_PK_Binding.col))
ctx.comment('Pickup height is ' +
str(round(pickup_height, 2)) + ' mm')
#if j!=0:
# rinse = False
move_vol_multi(m300,
reagent=Beads_PK_Binding,
source=Beads_PK_Binding.reagent_reservoir[
Beads_PK_Binding.col],
dest=work_destinations[i],
vol=transfer_vol,
x_offset_source=x_offset_source,
x_offset_dest=x_offset_dest,
pickup_height=pickup_height,
rinse=rinse,
avoid_droplet=False,
wait_time=2,
blow_out=True,
touch_tip=True,
drop_height=-1)
ctx.comment(' ')
ctx.comment('Mixing sample ')
custom_mix(m300,
Beads_PK_Binding,
location=work_destinations[i],
vol=Beads_PK_Binding.max_volume_allowed,
rounds=BEADS_NUM_MIXES,
blow_out=False,
mix_height=0,
offset=0,
wait_time=2,
two_thirds_mix_bottom=True)
# m300.move_to(work_destinations[i].top(0))
# m300.air_gap(Beads_PK_Binding.air_gap_vol_bottom) #air gap
if recycle_tip == True:
m300.return_tip()
else:
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
ctx.comment('Used tips in total: ' + str(tip_track['counts'][m300]))
###############################################################################
# STEP 1 TRANSFER BEADS + PK + Binding
########
###############################################################################
# STEP 2 TRANSFER WASH
########
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
wash_trips = math.ceil(Wash.reagent_volume / Wash.max_volume_allowed)
wash_volume = Wash.reagent_volume / wash_trips #136.66
wash_transfer_vol = []
for i in range(wash_trips):
wash_transfer_vol.append(wash_volume + Wash.disposal_volume)
x_offset_source = 0
x_offset_dest = 0
rinse = False
for i in range(num_cols):
ctx.comment("Column: " + str(i))
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(wash_transfer_vol):
ctx.comment('Aspirate from reservoir 1')
#if j!=0:
# rinse = False
move_vol_multi(m300,
reagent=Wash,
source=Wash.reagent_reservoir,
dest=wash_destinations[i],
vol=transfer_vol,
x_offset_source=x_offset_source,
x_offset_dest=x_offset_dest,
pickup_height=2,
rinse=rinse,
avoid_droplet=False,
wait_time=0,
blow_out=True,
touch_tip=True)
ctx.comment(' ')
if recycle_tip == True:
m300.return_tip()
else:
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
ctx.comment('Used tips in total: ' + str(tip_track['counts'][m300]))
###############################################################################
# STEP 2 TRANSFER WASH
########
###############################################################################
# STEP 3 TRANSFER ETHANOL
########
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
ethanol_trips = math.ceil(Ethanol.reagent_volume /
Ethanol.max_volume_allowed)
ethanol_volume = Ethanol.reagent_volume / ethanol_trips #136.66
ethanol_transfer_vol = []
for i in range(ethanol_trips):
ethanol_transfer_vol.append(ethanol_volume +
Ethanol.disposal_volume)
x_offset_source = 0
x_offset_dest = 0
rinse = False
for i in range(num_cols):
ctx.comment("Column: " + str(i))
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(ethanol_transfer_vol):
ctx.comment('Aspirate from reservoir 2')
#if j!=0:
# rinse = False
move_vol_multi(m300,
reagent=Ethanol,
source=Ethanol.reagent_reservoir,
dest=ethanol_destinations[i],
vol=transfer_vol,
x_offset_source=x_offset_source,
x_offset_dest=x_offset_dest,
pickup_height=2,
rinse=rinse,
avoid_droplet=False,
wait_time=0,
blow_out=True,
touch_tip=True)
if recycle_tip == True:
m300.return_tip()
else:
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
ctx.comment('Used tips in total: ' + str(tip_track['counts'][m300]))
###############################################################################
# STEP 3 TRANSFER ETHANOL
########
###############################################################################
# STEP 4 TRANSFER ELUTION
########
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
elution_trips = math.ceil(Elution.reagent_volume /
Elution.max_volume_allowed)
elution_volume = Elution.reagent_volume / elution_trips #136.66
elution_transfer_vol = []
for i in range(elution_trips):
elution_transfer_vol.append(elution_volume +
Elution.disposal_volume)
x_offset_source = 0
x_offset_dest = 0
rinse = False
for i in range(num_cols):
ctx.comment("Column: " + str(i))
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for j, transfer_vol in enumerate(elution_transfer_vol):
#Calculate pickup_height based on remaining volume and shape of container
[pickup_height,
change_col] = calc_height(Elution, multi_well_rack_area,
transfer_vol * 8)
ctx.comment('Aspirate from reservoir column: ' +
str(Elution.col))
ctx.comment('Pickup height is ' +
str(round(pickup_height, 2)) + ' mm')
#if j!=0:
# rinse = False
move_vol_multi(m300,
reagent=Elution,
source=Elution.reagent_reservoir[Elution.col],
dest=elution_destinations[i],
vol=transfer_vol,
x_offset_source=x_offset_source,
x_offset_dest=x_offset_dest,
pickup_height=pickup_height,
rinse=rinse,
avoid_droplet=False,
wait_time=0,
blow_out=False,
touch_tip=True)
ctx.comment(' ')
if recycle_tip == True:
m300.return_tip()
else:
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' + STEPS[STEP]['description'] +
' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
ctx.comment('Used tips in total: ' + str(tip_track['counts'][m300]))
###############################################################################
# STEP 4 TRANSFER ELUTION
########
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Homing robot')
ctx.comment('###############################################')
ctx.comment(' ')
ctx.home()
###############################################################################
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
# Light flash end of program
import os
#os.system('mpg123 /etc/audio/speaker-test.mp3')
for i in range(3):
ctx._hw_manager.hardware.set_lights(rails=False)
ctx._hw_manager.hardware.set_lights(button=(1, 0, 0))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails=True)
ctx._hw_manager.hardware.set_lights(button=(0, 0, 1))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=(0, 1, 0))
ctx.comment('Finished! \nMove deepwell plates Kingfisher robot.')
ctx.comment('Used tips 200uL in total: ' + str(tip_track['counts'][m300]))
ctx.comment('Used racks 200uL in total: ' +
str(tip_track['counts'][m300] / 96))
ctx.comment('Available 200uL tips: ' + str(tip_track['maxes'][m300]))
def run(ctx: protocol_api.ProtocolContext):
# Init protocol run
run = ProtocolRun(ctx)
# Define stesp
run.add_step(
description="Transfer PK+MS2 A6 - To AW_PLATE Single Slot1 -> Slot2") # 1
run.add_step(description="Transfer Beats 3 - 2 Multi and mix") # 2
# execute avaliaible steps
run.init_steps(steps)
##################################
# Tube rack
tube_rack = ctx.load_labware(
'opentrons_24_tuberack_nest_1.5ml_screwcap', 4)
# Destination plate SLOT 2
if(ctx.is_simulating()):
aw_slot = ctx.load_labware(
'opentrons_96_aluminumblock_generic_pcr_strip_200ul', 5)
else:
aw_slot = ctx.load_labware(
'axygen_96_wellplate_2000ul', 5)
aw_wells = aw_slot.wells()[:NUM_SAMPLES]
# Mount pippets and set racks
# Tipracks20_multi
tips20_1 = ctx.load_labware('opentrons_96_tiprack_20ul', 7)
tips20_2 = ctx.load_labware('opentrons_96_tiprack_20ul', 8)
run.mount_right_pip('p20_single_gen2', tip_racks=[tips20_1,tips20_2], capacity=20)
tips300 = ctx.load_labware('opentrons_96_filtertiprack_200ul', 7)
run.mount_right_pip('p20_single_gen2', tip_racks=[tips20_1,tips20_2], capacity=20)
############################################################################
# STEP 1: Transfer A6 - To AW_PLATE
############################################################################
if (run.next_step()):
run.set_pip("right") # single 20
volumen_move = 5
source = tube_rack.wells("A6")[0]
pkms2 = Reagent(
name='PK + MS2',
num_wells=1, # change with num samples
flow_rate_aspirate=0.75, # Original 0.5
flow_rate_dispense=3, # Original 1
reagent_reservoir_volume=vol_pkms2*(NUM_SAMPLES+1),
h_cono=4,
v_fondo=4 * math.pi * 4 ** 3 / 3
)
pkms2.set_positions([tube_rack.wells("A5"),tube_rack.wells("B5"),tube_rack.wells("B6")])
run.comment(pkms2.get_volumes_fill_print(),add_hash=True)
run.pick_up()
for dest in aw_wells:
pickup_height = run.calc_height(
liquid, 4.12*4.12*math.pi, vol_pkms2)
run.move_volume(reagent=liquid, source=source,
dest=pkms2.get_current_position(), vol=vol_pkms2, air_gap_vol=1,
pickup_height=pickup_height, disp_height=-10,
blow_out=True, post_dispense=True, post_dispense_vol=5)
run.drop_tip()
run.finish_step()
############################################################################
# STEP 2: Slot 3 -2 beats_PK AW
############################################################################
if (run.next_step()):
############################################################################
# Light flash end of program
run.set_pip("left") # p300 multi
volume = 275
beads = Reagent(name='Magnetic beads',
flow_rate_aspirate=0.5,
flow_rate_dispense=0.5,
flow_rate_dispense_mix=4,
flow_rate_aspirate_mix=4,
rinse=True,
delay=2,
reagent_reservoir_volume=vol_beads*(NUM_SAMPLES+1),
h_cono=1.95,
v_fondo=695,
rinse_loops=3)
beads.set_positions([tube_rack.wells("A6"),tube_rack.wells("B6")])
air_gap_vol = 5
disposal_height = -5
for destination in aw_wells:
volumes = beads.divide_volume(vol_beads,180)
for vol in volumes:
run.pick_up()
pickup_height = beads.calc_height(
beads, pool_area, vol, extra_volume=vol_min)
run.move_volume(reagent=beads, source=beads.get_current_position(),
dest=destination, vol=vol, air_gap_vol=air_gap_vol,
pickup_height=pickup_height, disp_height=disposal_height,
rinse=True, blow_out=True)
run.custom_mix(beads, location=destination, vol=vol/2,
rounds=3, blow_out=True, mix_height=0)
run.drop_tip()
run.finish_step()
run.log_steps_time()
run.blink()
for c in robot.commands():
ctx.comment(c)
ctx.comment('Finished! \nMove plate to PCR')
def run(ctx: protocol_api.ProtocolContext):
STEP = 0
STEPS = { # Dictionary with STEP activation, description and times
1: {
'Execute': True,
'description': 'Dispensar Antibioticos'
},
2: {
'Execute': True,
'description': 'Dispensar caldo y mezclar '
}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
if not ctx.is_simulating():
folder_path = '/var/lib/jupyter/notebooks/' + run_id
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/time_log.txt'
# Define Reagents as objects with their properties
#Define Reagents as objects with their properties
class Reagent:
def __init__(self,
name,
flow_rate_aspirate,
flow_rate_dispense,
flow_rate_aspirate_mix,
flow_rate_dispense_mix,
air_gap_vol_bottom,
air_gap_vol_top,
disposal_volume,
rinse,
max_volume_allowed,
reagent_volume,
reagent_reservoir_volume,
num_wells,
h_cono,
v_fondo,
tip_recycling='none',
dead_vol=DEFAULT_DEAD_VOL):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.flow_rate_aspirate_mix = flow_rate_aspirate_mix
self.flow_rate_dispense_mix = flow_rate_dispense_mix
self.air_gap_vol_bottom = air_gap_vol_bottom
self.air_gap_vol_top = air_gap_vol_top
self.disposal_volume = disposal_volume
self.rinse = bool(rinse)
self.max_volume_allowed = max_volume_allowed
self.reagent_volume = reagent_volume
self.reagent_reservoir_volume = reagent_reservoir_volume
self.num_wells = num_wells
self.col = 0
self.vol_well = 0
self.h_cono = h_cono
self.v_cono = v_fondo
self.tip_recycling = tip_recycling
self.dead_vol = dead_vol
self.vol_well_original = (reagent_reservoir_volume / num_wells
) + dead_vol if num_wells > 0 else 0
# Reagents and their characteristics
Samples = Reagent(
name='Antibioticos',
flow_rate_aspirate=3,
flow_rate_dispense=3,
flow_rate_aspirate_mix=1,
flow_rate_dispense_mix=1,
air_gap_vol_bottom=5,
air_gap_vol_top=0,
disposal_volume=0,
rinse=True,
max_volume_allowed=180,
reagent_volume=VOLUME_SAMPLE,
reagent_reservoir_volume=NUM_REAL_SAMPLES * VOLUME_SAMPLE * 1.1,
num_wells=math.ceil(NUM_REAL_SAMPLES * VOLUME_SAMPLE * 1.1 / 11500),
h_cono=1.95,
v_fondo=695) #1.95 * multi_well_rack_area / 2, #Prismatic
Caldo = Reagent(
name='Caldo ',
flow_rate_aspirate=2,
flow_rate_dispense=2,
flow_rate_aspirate_mix=1,
flow_rate_dispense_mix=1,
air_gap_vol_bottom=6,
air_gap_vol_top=0,
disposal_volume=0,
rinse=True,
max_volume_allowed=180,
reagent_volume=VOLUME_SAMPLE,
reagent_reservoir_volume=NUM_REAL_SAMPLES * VOLUME_SAMPLE * 1.1,
num_wells=math.ceil(NUM_REAL_SAMPLES * VOLUME_SAMPLE * 1.1 / 11500),
h_cono=1.95,
v_fondo=695) #1.95 * multi_well_rack_area / 2, #Prismatic
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('VALORES DE VARIABLES')
ctx.comment(' ')
ctx.comment('Número Antibióticos: ' + str(NUM_SAMPLE_ANTIBIOTIC) + ' (' +
str(NUM_FINAL_PLATES) + ' placas)')
ctx.comment('Número de muestras: ' + str(NUM_REAL_SAMPLES) + ' (' +
str(math.ceil(NUM_REAL_SAMPLES / 8)) + ' columnas)')
ctx.comment('Número de mezclas: ' + str(NUM_DILUTION_MIXES))
ctx.comment(' ')
ctx.comment('Volumen de muestra a mover al deepwell: ' +
str(VOLUME_SAMPLE) + ' ul')
ctx.comment(' ')
ctx.comment('Número de mezclas en la muestra: ' + str(TUBE_NUM_MIXES))
ctx.comment(' ')
ctx.comment('Repeticiones del sonido final: ' + str(SOUND_NUM_PLAYS))
ctx.comment('Foto-sensible: ' + str(PHOTOSENSITIVE))
ctx.comment(' ')
##################
# Custom functions
def move_multichanel_caldo(dest):
if not m300.hw_pipette['has_tip']:
pick_up_multi(m300)
caldo_trips = math.floor(Caldo.max_volume_allowed /
Caldo.reagent_volume)
caldo_volume = Caldo.reagent_volume * caldo_trips #136.66
Caldo.max_volume_allowed = caldo_volume
caldo_transfer_vol = []
x_offset_source = 0
x_offset_dest = 0
rinse = False # Original: True
first_mix_done = False
actual_pipette_vol = 0
for i in range(num_cols_caldo):
ctx.comment("Column: " + str(i))
#Calculate pickup_height based on remaining volume and shape of container
ctx.comment('Aspirate from reservoir column: ' + str(Caldo.col))
if actual_pipette_vol >= VOLUME_SAMPLE:
recharge = False
else:
actual_pipette_vol = Caldo.max_volume_allowed
recharge = True
move_vol_multi(m300,
reagent=Caldo,
source=Caldo.reagent_reservoir,
dest=dest[i],
vol=Caldo.reagent_volume,
x_offset_source=x_offset_source,
x_offset_dest=x_offset_dest,
pickup_height=0,
rinse=rinse,
wait_time=0.5,
blow_out=False,
touch_tip=True,
drop_height=-3,
recharge=recharge)
actual_pipette_vol = actual_pipette_vol - VOLUME_SAMPLE
m300.blow_out(Caldo.reagent_reservoir.top(z=0))
ctx.delay(seconds=2, msg='Waiting for ' + str(1) + ' seconds.')
m300.move_to(Caldo.reagent_reservoir.bottom(2))
m300.blow_out(Caldo.reagent_reservoir.top(z=0))
def move_multichanel_mezcla(origin, dest, pickup_height=2, air_gap_vol=5):
x_offset_source = 0
x_offset_dest = 0
first_mix_done = False
if not m300.hw_pipette['has_tip']:
pick_up(m300)
for i in range(num_cols_caldo):
ctx.comment("Column: " + str(i))
ctx.comment('Aspirate from reservoir column: ' + str(Caldo.col))
move_vol_multi(m300,
reagent=Caldo,
source=origin[i],
dest=dest[i],
vol=Caldo.reagent_volume,
multitripCount=1,
x_offset_source=x_offset_source,
x_offset_dest=x_offset_dest,
pickup_height=pickup_height,
rinse=False,
rinseEnd=True,
wait_time=0.5,
blow_out=False,
touch_tip=False,
drop_height=-1,
rate_multiplier=0.1)
# Aspirar 50 ul de la última columna para igualar volúmenes
m300.aspirate(Caldo.reagent_volume,
location=dest[num_cols_caldo -
1].bottom(z=pickup_height),
rate=Caldo.flow_rate_aspirate)
if air_gap_vol > 0:
m300.aspirate(air_gap_vol,
dest[num_cols_caldo - 1].top(z=0),
rate=Caldo.flow_rate_aspirate) # air gap
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 8
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol,
x_offset, pickup_height, rinse, disp_height,
blow_out, touch_tip):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# SOURCE
s = source.bottom(pickup_height).move(Point(x=x_offset[0]))
pipet.aspirate(vol, s,
rate=reagent.flow_rate_aspirate) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol,
source.top(z=-2),
rate=reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z=disp_height).move(Point(x=x_offset[1]))
pipet.dispense(vol + air_gap_vol,
drop,
rate=reagent.flow_rate_dispense) # dispense all
#ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
if blow_out == True:
pipet.blow_out(dest.top(z=disp_height))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-10)
if air_gap_vol != 0:
pipet.air_gap(air_gap_vol, height=disp_height) #air gap
def move_vol_multi(pipet,
reagent,
source,
dest,
vol,
x_offset_source,
x_offset_dest,
pickup_height,
rinse,
wait_time,
blow_out,
touch_tip=False,
drop_height=-5,
dispense_bottom_air_gap_before=False,
rinseEnd=False,
recharge=True,
multitripCount=3,
rate_multiplier=1):
# Rinse before aspirating
if rinse == True:
#pipet.aspirate(air_gap_vol_top, location = source.top(z = -5), rate = reagent.flow_rate_aspirate) #air gap
custom_mix(pipet,
reagent,
location=source,
vol=vol * .6,
rounds=NUM_DILUTION_MIXES,
blow_out=False,
mix_height=pickup_height,
offset=0)
#pipet.dispense(air_gap_vol_top, location = source.top(z = -5), rate = reagent.flow_rate_dispense)
# SOURCE
if recharge == True:
#if dispense_bottom_air_gap_before and reagent.air_gap_vol_bottom:
# pipet.dispense(reagent.air_gap_vol_bottom, source.top(z = -2), rate = reagent.flow_rate_dispense)
if reagent.air_gap_vol_top != 0: #If there is air_gap_vol, switch pipette to slow speed
pipet.move_to(source.top(z=0))
pipet.air_gap(reagent.air_gap_vol_top) #air gap
#pipet.aspirate(reagent.air_gap_vol_top, source.top(z = -5), rate = reagent.flow_rate_aspirate) #air gap
pipet.blow_out(source.top(z=drop_height))
s = source.bottom(pickup_height).move(Point(x=x_offset_source))
pipet.aspirate(vol * multitripCount,
s,
rate=reagent.flow_rate_aspirate *
rate_multiplier) # aspirate liquid
if reagent.air_gap_vol_bottom != 0: #If there is air_gap_vol, switch pipette to slow speed
pipet.air_gap(reagent.air_gap_vol_bottom, height=0) #air gap
if wait_time != 0:
ctx.delay(seconds=wait_time,
msg='Waiting for ' + str(wait_time) + ' seconds.')
# GO TO DESTINATION
d = dest.top(z=drop_height).move(Point(x=x_offset_dest))
pipet.dispense(vol + reagent.air_gap_vol_bottom,
d,
rate=reagent.flow_rate_dispense * rate_multiplier)
if wait_time != 0:
ctx.delay(seconds=wait_time,
msg='Waiting for ' + str(wait_time) + ' seconds.')
if reagent.air_gap_vol_top != 0:
pipet.dispense(reagent.air_gap_vol_top,
dest.top(z=0),
rate=reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(dest.top(z=drop_height))
if touch_tip == True:
pipet.touch_tip(speed=20, v_offset=-3, radius=0.7)
# Rinse after aspirating
if rinseEnd == True:
#pipet.aspirate(air_gap_vol_top, location = source.top(z = -5), rate = reagent.flow_rate_aspirate) #air gap
custom_mix(pipet,
reagent,
location=dest,
vol=vol * .6,
rounds=2,
blow_out=False,
mix_height=pickup_height,
offset=0)
pipet.blow_out(dest.top(z=drop_height))
#pipet.dispense(air_gap_vol_top, location = source.top(z = -5), rate = reagent.flow_rate_dispense)
if reagent.air_gap_vol_bottom != 0 and rinseEnd == False:
pipet.move_to(dest.top(z=0))
pipet.air_gap(reagent.air_gap_vol_bottom) #air gap
#pipet.aspirate(air_gap_vol_bottom, dest.top(z = 0),rate = reagent.flow_rate_aspirate) #air gap
def custom_mix(pipet,
reagent,
location,
vol,
rounds,
blow_out,
mix_height,
offset,
wait_time=0,
drop_height=-1,
two_thirds_mix_bottom=False):
'''
Function for mix in the same location a certain number of rounds. Blow out optional. Offset
can set to 0 or a higher/lower value which indicates the lateral movement
'''
if mix_height <= 0:
mix_height = 1
pipet.aspirate(1,
location=location.bottom(z=mix_height),
rate=reagent.flow_rate_aspirate_mix)
for i in range(rounds):
pipet.aspirate(vol,
location=location.bottom(z=mix_height),
rate=reagent.flow_rate_aspirate_mix)
if two_thirds_mix_bottom and i < ((rounds / 3) * 2):
pipet.dispense(vol,
location=location.bottom(z=5).move(
Point(x=offset)),
rate=reagent.flow_rate_dispense_mix)
else:
pipet.dispense(vol,
location=location.top(z=drop_height).move(
Point(x=offset)),
rate=reagent.flow_rate_dispense_mix)
pipet.dispense(1,
location=location.bottom(z=mix_height),
rate=reagent.flow_rate_dispense_mix)
if blow_out == True:
pipet.blow_out(location.top(z=-2)) # Blow out
if wait_time != 0:
ctx.delay(seconds=wait_time,
msg='Waiting for ' + str(wait_time) + ' seconds.')
def generate_atibiotic_source(source_plate):
result = []
'''
for i in range (NUM_FINAL_PLATES):
result += source_plate.rows()[i][0]
'''
result = source_plate.rows()[:NUM_FINAL_PLATES][0]
return result
def generate_antibiotic_dest(dest_plates):
'''
Concatenate cols from all destination plates
'''
result = []
#result = dest_plates[0].columns()[0][0] + dest_plates[1].columns()[0] + dest_plates[2].columns()[0]
for i in range(NUM_FINAL_PLATES):
result += [dest_plates[i].columns()[0][0]]
return result
def calc_height(reagent,
cross_section_area,
aspirate_volume,
min_height=0.4):
nonlocal ctx
ctx.comment('Remaining volume ' + str(reagent.vol_well) +
'< needed volume ' + str(aspirate_volume) + '?')
if (reagent.vol_well - reagent.dead_vol) < aspirate_volume:
ctx.comment('Next column should be picked')
ctx.comment('Previous to change: ' + str(reagent.col))
# column selector position; intialize to required number
reagent.col = reagent.col + 1
ctx.comment(str('After change: ' + str(reagent.col)))
reagent.vol_well = reagent.vol_well_original
ctx.comment('New volume:' + str(reagent.vol_well))
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
#- reagent.h_cono
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Remaining volume:' + str(reagent.vol_well))
if height < min_height:
height = min_height
col_change = True
else:
height = (reagent.vol_well - aspirate_volume -
reagent.v_cono) / cross_section_area
reagent.vol_well = reagent.vol_well - aspirate_volume
ctx.comment('Calculated height is ' + str(height))
if height < min_height:
height = min_height
ctx.comment('Used height is ' + str(height))
col_change = False
return height, col_change
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) +
'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
pip.pick_up_tip()
def pick_up_multi(pip):
nonlocal tip_track
#if not ctx.is_simulating():
if tip_track['counts'][pip] >= tip_track['maxes'][pip]:
for i in range(3):
ctx._hw_manager.hardware.set_lights(rails=False)
ctx._hw_manager.hardware.set_lights(button=(1, 0, 0))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(rails=True)
ctx._hw_manager.hardware.set_lights(button=(0, 0, 1))
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=(0, 1, 0))
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
tip_track['num_refills'][pip] += 1
pip.pick_up_tip()
def run_quiet_process(command):
subprocess.check_output('{} &> /dev/null'.format(command), shell=True)
def play_sound(filename):
print('Speaker')
print('Next\t--> CTRL-C')
try:
run_quiet_process('mpg123 {}'.format(path_sounds + filename +
'.mp3'))
run_quiet_process('mpg123 {}'.format(path_sounds + sonido_defecto))
run_quiet_process('mpg123 {}'.format(path_sounds + filename +
'.mp3'))
except KeyboardInterrupt:
pass
print()
def start_run():
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Empezando protocolo')
if PHOTOSENSITIVE == False:
ctx._hw_manager.hardware.set_lights(button=True, rails=True)
else:
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
now = datetime.now()
# dd/mm/YY H:M:S
start_time = now.strftime("%Y/%m/%d %H:%M:%S")
return start_time
def finish_run(switch_off_lights=False):
ctx.comment('###############################################')
ctx.comment('Protocolo finalizado')
ctx.comment(' ')
#Set light color to blue
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
now = datetime.now()
# dd/mm/YY H:M:S
finish_time = now.strftime("%Y/%m/%d %H:%M:%S")
if PHOTOSENSITIVE == False:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button=False, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=True)
time.sleep(0.3)
else:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button=False, rails=False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
time.sleep(0.3)
if switch_off_lights:
ctx._hw_manager.hardware.set_lights(button=True, rails=False)
used_tips_300 = tip_track['num_refills'][m300] * 96 * len(
m300.tip_racks) + tip_track['counts'][m300]
ctx.comment('Puntas de 300 ul utilizadas: ' + str(used_tips_300) +
' (' + str(round(used_tips_300 / 96, 2)) + ' caja(s))')
ctx.comment('###############################################')
if not ctx.is_simulating():
for i in range(SOUND_NUM_PLAYS):
if i > 0:
time.sleep(60)
play_sound('finished_process_esp')
return finish_time
def validate_parameters():
result = True
return result
####################################
# load labware and modules
####################################
# Load Sample racks
source_antibiotic = ctx.load_labware('nest_96_wellplate_2ml_deep', '8',
'NEST 96 Deepwell Plate 2mL')
'''
ctx.load_labware(
'opentrons_24_tuberack_eppendorf_2ml_safelock_snapcap', '8',
'Opentrons 24 Tuberack Eppendorf 2ml Safelock Snapcap')
'''
source_caldo = ctx.load_labware('nest_1_reservoir_195ml', '1',
'NEST 1 Reservoir 195ul')
##################################
# Destination plate
# Destination
dest_plate1 = ctx.load_labware('nest_96_wellplate_200ul_flat', '3',
'NEST 96 Well Plate 200ul 1')
dest_plate2 = ctx.load_labware('nest_96_wellplate_200ul_flat', '5',
'NEST 96 Well Plate 200ul 2')
dest_plate3 = ctx.load_labware('nest_96_wellplate_200ul_flat', '7',
'NEST 96 Well Plate 200ul 3')
####################################
# Load tip_racks
tips200 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['10']
]
################################################################################
# setup samples and destinations
num_destination_plates = NUM_FINAL_PLATES
#sample_antibiotic = source_antibiotic.wells()[:NUM_SAMPLE_ANTIBIOTIC]
sample_antibiotic = generate_atibiotic_source(source_antibiotic)
Caldo.reagent_reservoir = source_caldo.wells()[0]
destinations_antibiotic = generate_antibiotic_dest(
[dest_plate1, dest_plate2, dest_plate3]) #[:NUM_SAMPLE_ANTIBIOTIC]
destinations1 = dest_plate1.rows()[0][1:]
destinations2 = dest_plate2.rows()[0][1:]
destinations3 = dest_plate3.rows()[0][1:]
sourceMix1 = dest_plate1.rows()[0][:11]
sourceMix2 = dest_plate2.rows()[0][:11]
sourceMix3 = dest_plate3.rows()[0][:11]
destinationsMix1 = dest_plate1.rows()[0][1:12]
destinationsMix2 = dest_plate2.rows()[0][1:12]
destinationsMix3 = dest_plate3.rows()[0][1:12]
m300 = ctx.load_instrument('p300_multi_gen2', 'right',
tip_racks=tips200) # load P300 pipette
# used tip counter and set maximum tips available
tip_track = {
'counts': {
m300: 0
},
'maxes': {
m300: 96 * len(m300.tip_racks)
},
'num_refills': {
m300: 0
},
'tips': {
m300: [tip for rack in tips200 for tip in rack.rows()[0]]
}
}
if validate_parameters():
start_run()
############################################################################
# STEP 1: Dispensación de Atnibiótico
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'])
ctx.comment('###############################################')
start = datetime.now()
for s, d in zip(sample_antibiotic, destinations_antibiotic):
# Mix the sample BEFORE dispensing
if not m300.hw_pipette['has_tip']:
pick_up(m300)
move_vol_multichannel(m300,
reagent=Samples,
source=s,
dest=d,
vol=VOLUME_ANTBIOTIC,
air_gap_vol=air_gap_vol_sample,
x_offset=x_offset,
pickup_height=3,
rinse=Samples.rinse,
disp_height=0,
blow_out=True,
touch_tip=False)
m300.drop_tip(home_after=False)
tip_track['counts'][m300] += 1
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' +
str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: Dilución de antibiótico sobre el caldo
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'])
ctx.comment('###############################################')
start = datetime.now()
if num_destination_plates >= 1:
move_multichanel_caldo(destinations1)
move_multichanel_mezcla(sourceMix1, destinationsMix1)
if num_destination_plates >= 2:
move_multichanel_caldo(destinations2)
move_multichanel_mezcla(sourceMix2, destinationsMix2)
if num_destination_plates >= 3:
move_multichanel_caldo(destinations3)
move_multichanel_mezcla(sourceMix3, destinationsMix3)
# Time statistics
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' +
str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write(
'STEP\texecution\tdescription\twait_time\texecution_time\n'
)
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
finish_run(switch_off_lights)
def run(ctx: protocol_api.ProtocolContext):
# load labware
ic_pk = ctx.load_labware(
'opentrons_24_aluminumblock_nest_2ml_snapcap', '9',
'chilled tubeblock for internal control and proteinase K (strip 1)'
).wells()[0]
source_racks = [
ctx.load_labware(
'opentrons_24_tuberack_eppendorf_2ml_safelock_snapcap', slot,
'source tuberack ' + str(i + 1))
for i, slot in enumerate(['1', '2', '3', '4'])
]
dest_plate = ctx.load_labware('nest_96_wellplate_2ml_deep', '8',
'96-deepwell sample plate')
binding_buffer = ctx.load_labware(
'opentrons_6_tuberack_falcon_50ml_conical', '11',
'50ml tuberack for binding buffer (tube B1)').wells('B1')
# binding_buffer = ctx.load_labware(
# 'biorad_96_wellplate_200ul_pcr', '7',
# '50ml tuberack for lysis buffer + PK (tube A1)').wells()[:1]
tipracks1000 = [
ctx.load_labware('opentrons_96_filtertiprack_1000ul', slot,
'1000µl filter tiprack') for slot in ['10', '7']
]
tipracks20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', '6',
'20µl filter tiprack')
]
# load pipette
s20 = ctx.load_instrument('p20_single_gen2', 'left', tip_racks=tipracks20)
p1000 = ctx.load_instrument('p1000_single_gen2',
'right',
tip_racks=tipracks1000)
# setup samples
sources = [well for rack in source_racks
for well in rack.wells()][:NUM_SAMPLES]
dests_single = dest_plate.wells()[:NUM_SAMPLES]
num_cols = math.ceil(NUM_SAMPLES / 8)
dests_multi = dest_plate.rows()[0][:num_cols]
tip_log = {'count': {}}
folder_path = '/data/A'
tip_file_path = folder_path + '/tip_log.json'
if TIP_TRACK and not ctx.is_simulating():
if os.path.isfile(tip_file_path):
with open(tip_file_path) as json_file:
data = json.load(json_file)
if 'tips1000' in data:
tip_log['count'][p1000] = data['tips1000']
else:
tip_log['count'][p1000] = 0
if 'tips20' in data:
tip_log['count'][s20] = data['tips20']
else:
tip_log['count'][s20] = 0
else:
tip_log['count'] = {p1000: 0, s20: 0}
tip_log['tips'] = {
p1000: [tip for rack in tipracks1000 for tip in rack.wells()],
#s20: [tip for rack in tipracks20 for tip in rack.rows()[0]]
s20: [tip for rack in tipracks20 for tip in rack.wells()]
}
tip_log['max'] = {pip: len(tip_log['tips'][pip]) for pip in [p1000, s20]}
def pick_up(pip):
nonlocal tip_log
if tip_log['count'][pip] == tip_log['max'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_log['count'][pip] = 0
pip.pick_up_tip(tip_log['tips'][pip][tip_log['count'][pip]])
tip_log['count'][pip] += 1
heights = {tube: TUBE50_VOlUME * 1 for tube in binding_buffer}
radius = (binding_buffer[0].diameter) / 2
min_h = 5
def h_track(vol, tube):
nonlocal heights
dh = vol / (math.pi * (radius**2))
if heights[tube] - dh > min_h:
heights[tube] = heights[tube] - dh
else:
heights[tube] = min_h # stop 5mm short of the bottom
return heights[tube]
p1000.flow_rate.aspirate = 50
p1000.flow_rate.dispense = 60
p1000.flow_rate.blow_out = 100
# transfer internal control + proteinase K
pick_up(s20)
for d in dests_single:
s20.dispense(10, ic_pk.bottom(2))
s20.transfer(ICPK_VOlUME,
ic_pk.bottom(2),
d.bottom(2),
air_gap=5,
new_tip='never')
s20.air_gap(5)
s20.drop_tip()
# transfer binding buffer and mix
pick_up(p1000)
for i, (s, d) in enumerate(zip(sources, dests_single)):
source = binding_buffer[
i //
96] # 1 tube of binding buffer can accommodate all samples here
h = h_track(275, source)
# custom mix
p1000.flow_rate.aspirate = 100
p1000.flow_rate.dispense = 100
p1000.dispense(500, source.bottom(h + 20))
for _ in range(4):
# p1000.air_gap(500)
p1000.aspirate(500, source.bottom(h))
p1000.dispense(500, source.bottom(h + 20))
# p1000.transfer(BB_VOLUME, source.bottom(h), d.bottom(5), air_gap=100,
# new_tip='never')
p1000.flow_rate.aspirate = 50
p1000.flow_rate.dispense = 100
p1000.aspirate(BB_VOLUME, source.bottom(h))
p1000.air_gap(10)
p1000.dispense(BB_VOLUME + 100, d.bottom(10))
p1000.air_gap(10)
p1000.drop_tip()
ctx.comment('Move deepwell plate (slot 4) to Station B for RNA \
extraction.')
# track final used tip
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
data = {
'tips1000': tip_log['count'][p1000],
'tips20': tip_log['count'][s20]
}
with open(tip_file_path, 'w') as outfile:
json.dump(data, outfile)
def run(ctx: protocol_api.ProtocolContext):
# Define the STEPS of the protocol
STEP = 0
STEPS = { # Dictionary with STEP activation, description, and times
1: {'Execute': HYDRATATE, 'description': 'Hidratate'},
2: {'Execute': True, 'description': 'Transfer samples'}
}
for s in STEPS: # Create an empty wait_time
if 'wait_time' not in STEPS[s]:
STEPS[s]['wait_time'] = 0
#Folder and file_path for log time
folder_path = '/var/lib/jupyter/notebooks' + run_id
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
file_path = folder_path + '/time_log.txt'
# Define Reagents as objects with their properties
class Reagent:
def __init__(self, name, flow_rate_aspirate, flow_rate_dispense, rinse,
reagent_reservoir_volume, delay, num_wells):
self.name = name
self.flow_rate_aspirate = flow_rate_aspirate
self.flow_rate_dispense = flow_rate_dispense
self.rinse = bool(rinse)
self.reagent_reservoir_volume = reagent_reservoir_volume
self.delay = delay
self.num_wells = num_wells
self.vol_well = 0
self.unused = []
self.vol_well_original = reagent_reservoir_volume / num_wells
# Reagents and their characteristics
Hydr = Reagent(name = 'Hydr',
rinse = False,
flow_rate_aspirate = 3,
flow_rate_dispense = 3,
reagent_reservoir_volume = 1800,
num_wells = 1,
delay = 0
)
Samples = Reagent(name = 'Samples',
rinse = False,
flow_rate_aspirate = 1,
flow_rate_dispense = 1,
reagent_reservoir_volume = 50,
delay = 0,
num_wells = NUM_SAMPLES
)
Hydr.vol_well = Hydr.vol_well_original
Samples.vol_well = Samples.vol_well_original
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('VALORES DE VARIABLES')
ctx.comment(' ')
ctx.comment('Número de muestras: ' + str(NUM_SAMPLES) + ' las dos primeras son controles.')
ctx.comment(' ')
ctx.comment('Hidratar muestras: ' + str(HYDRATATE))
if HYDRATATE:
ctx.comment('Volumen de Hidratante por muestra: ' + str(HYDR_VOL_PER_SAMPLE) + ' uL')
ctx.comment(' ')
ctx.comment('Volumen de muestra: ' + str(VOLUME_SAMPLE) + ' uL')
ctx.comment(' ')
ctx.comment('Foto-sensible: ' + str(PHOTOSENSITIVE))
ctx.comment('Repeticiones del sonido final: ' + str(SOUND_NUM_PLAYS))
ctx.comment(' ')
##################
# Custom functions
def divide_destinations(l, n):
# Divide the list of destinations in size n lists.
for i in range(0, len(l), n):
yield l[i:i + n]
def distribute_custom(pipette, volume, src, dest, waste_pool, pickup_height, extra_dispensal, dest_x_offset, disp_height = 0, touch_tip = False, num_shakes = 0):
pipette.aspirate((len(dest) * volume) + extra_dispensal, src.bottom(pickup_height))
if touch_tip :
pipette.touch_tip(speed = 20, v_offset = -5)
for d in dest:
drop = d.top(z = disp_height)
pipette.dispense(volume, drop)
shake_pipet(pipette, rounds = num_shakes, v_offset = disp_height)
try:
pipette.blow_out(waste_pool.wells()[0].bottom(pickup_height + 3))
except:
pipette.blow_out(waste_pool.bottom(pickup_height + 3))
return (len(dest) * volume)
def shake_pipet (pipet, rounds = 2, speed = 100, v_offset = 0):
ctx.comment("Shaking " + str(rounds) + " rounds.")
for i in range(rounds):
pipet.touch_tip(speed = speed, radius = 0.1, v_offset = v_offset)
def move_vol_multichannel(pipet, reagent, source, dest, vol, air_gap_vol, x_offset,
pickup_height, rinse, disp_height, blow_out, touch_tip, num_shakes = 0):
'''
x_offset: list with two values. x_offset in source and x_offset in destination i.e. [-1,1]
pickup_height: height from bottom where volume
rinse: if True it will do 2 rounds of aspirate and dispense before the tranfer
disp_height: dispense height; by default it's close to the top (z=-2), but in case it is needed it can be lowered
blow_out, touch_tip: if True they will be done after dispensing
'''
# Rinse before aspirating
if rinse == True:
custom_mix(pipet, reagent, location = source, vol = vol,
rounds = 2, blow_out = True, mix_height = 0,
x_offset = x_offset)
# SOURCE
s = source.bottom(pickup_height).move(Point(x = x_offset[0]))
pipet.aspirate(vol, s) # aspirate liquid
if air_gap_vol != 0: # If there is air_gap_vol, switch pipette to slow speed
pipet.aspirate(air_gap_vol, source.top(z = -2),
rate = reagent.flow_rate_aspirate) # air gap
# GO TO DESTINATION
drop = dest.top(z = disp_height).move(Point(x = x_offset[1]))
pipet.dispense(vol + air_gap_vol, drop,
rate = reagent.flow_rate_dispense) # dispense all
ctx.delay(seconds = reagent.delay) # pause for x seconds depending on reagent
shake_pipet(pipet, rounds = num_shakes, v_offset = disp_height)
if blow_out == True:
pipet.blow_out(dest.top(z = -10))
if touch_tip == True:
pipet.touch_tip(speed = 20, v_offset = -10, radius = 0.5)
def custom_mix(pipet, reagent, location, vol, rounds, blow_out, mix_height,
x_offset, source_height = 3):
'''
Function for mixing a given [vol] in the same [location] a x number of [rounds].
blow_out: Blow out optional [True,False]
x_offset = [source, destination]
source_height: height from bottom to aspirate
mix_height: height from bottom to dispense
'''
if mix_height <= 0:
mix_height = 3
pipet.aspirate(1, location = location.bottom(
z = source_height).move(Point(x = x_offset[0])), rate = reagent.flow_rate_aspirate)
for _ in range(rounds):
pipet.aspirate(vol, location = location.bottom(
z = source_height).move(Point(x = x_offset[0])), rate = reagent.flow_rate_aspirate)
pipet.dispense(vol, location = location.bottom(
z = mix_height).move(Point(x = x_offset[1])), rate = reagent.flow_rate_dispense)
pipet.dispense(1, location = location.bottom(
z = mix_height).move(Point(x = x_offset[1])), rate = reagent.flow_rate_dispense)
if blow_out == True:
pipet.blow_out(location.top(z = -2)) # Blow out
def run_quiet_process(command):
subprocess.check_output('{} &> /dev/null'.format(command), shell=True)
def play_sound(filename):
print('Speaker')
print('Next\t--> CTRL-C')
try:
run_quiet_process('mpg123 {}'.format(path_sounds + filename + '.mp3'))
except KeyboardInterrupt:
pass
print()
def finish_run(switch_off_lights = False):
ctx.comment('###############################################')
ctx.comment('Protocolo finalizado')
ctx.comment(' ')
#Set light color to blue
ctx._hw_manager.hardware.set_lights(button = True, rails = False)
now = datetime.now()
# dd/mm/YY H:M:S
finish_time = now.strftime("%Y/%m/%d %H:%M:%S")
if PHOTOSENSITIVE==False:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button = False, rails = False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button = True, rails = True)
time.sleep(0.3)
else:
for i in range(10):
ctx._hw_manager.hardware.set_lights(button = False, rails = False)
time.sleep(0.3)
ctx._hw_manager.hardware.set_lights(button = True, rails = False)
time.sleep(0.3)
if switch_off_lights:
ctx._hw_manager.hardware.set_lights(button = True, rails = False)
ctx.comment('Puntas de 20 uL utilizadas: ' + str(tip_track['counts'][m20]) + ' (' + str(round(tip_track['counts'][m20] / 96, 2)) + ' caja(s))')
ctx.comment('###############################################')
if not ctx.is_simulating():
for i in range(SOUND_NUM_PLAYS):
if i > 0:
time.sleep(60)
play_sound('finalizado')
return finish_time
####################################
# load labware and modules
####################################
####################################
# 24 well rack
tuberack = ctx.load_labware(
'opentrons_24_aluminumblock_generic_2ml_screwcap', '8',
'Opentrons 24 Well Aluminum Block with Generic 2 mL Screwcap')
##################################
# Sample plate - comes from B
source_plate = ctx.load_labware(
'biorad_96_wellplate_200ul_pcr', '3',
'Bio-Rad 96 Well Plate 200 µL PCR')
##################################
# qPCR plate - final plate, goes to PCR
qpcr_plate = ctx.load_labware(
'opentrons_96_aluminumblock_generic_pcr_strip_200ul', '6',
'Opentrons 96 Well Aluminum Block with Generic PCR Strip 200 µL')
##################################
# Load Tipracks
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', slot)
for slot in ['2']
]
tips200 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['5']
]
################################################################################
# Declare which reagents are in each reservoir as well as deepwell and elution plate
Hydr.reagent_reservoir = tuberack.rows()[0][0] # A1
# setup up sample sources and destinations
samples = source_plate.rows()[0][:num_cols]
pcr_wells = qpcr_plate.wells()[:NUM_SAMPLES]
pcr_wells_samples = qpcr_plate.rows()[0][:num_cols]
tipCols = tips20[0].rows()[0][:num_cols]
# Divide destination wells in small groups for P300 pipette
dests = list(divide_destinations(pcr_wells, size_transfer))
# pipettes
m20 = ctx.load_instrument(
'p20_multi_gen2', mount = 'right',
tip_racks = tips20) # load m20 pipette
p300 = ctx.load_instrument(
'p300_single_gen2', mount = 'left', tip_racks = tips200)
# used tip counter and set maximum tips available
tip_track = {
'counts': {p300: 0,
m20: 0},
'maxes': {p300: 96 * len(p300.tip_racks),
m20: 96 * len(m20.tip_racks)}
}
##########
# pick up tip and if there is none left, prompt user for a new rack
def pick_up(pip):
nonlocal tip_track
if not ctx.is_simulating():
if tip_track['counts'][pip] == tip_track['maxes'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) + 'µl tipracks before \
resuming.')
pip.reset_tipracks()
tip_track['counts'][pip] = 0
if not pip.hw_pipette['has_tip']:
pip.pick_up_tip()
##########
############################################################################
# STEP 1: HIDRATATE
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step '+str(STEP)+': '+STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
pick_up(p300)
used_vol = []
for dest in dests:
aspirate_volume = HYDR_VOL_PER_SAMPLE * len(dest) + extra_dispensal
used_vol_temp = distribute_custom(p300, volume = HYDR_VOL_PER_SAMPLE,
src = Hydr.reagent_reservoir, dest = dest, touch_tip = False,
waste_pool = Hydr.reagent_reservoir, pickup_height = 0.2,
extra_dispensal = extra_dispensal, dest_x_offset = 0,
disp_height = -15, num_shakes = 1)
used_vol.append(used_vol_temp)
p300.drop_tip(home_after = False)
tip_track['counts'][p300] += 1
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
############################################################################
# STEP 2: TRANSFER SAMPLES
############################################################################
STEP += 1
if STEPS[STEP]['Execute'] == True:
start = datetime.now()
ctx.comment(' ')
ctx.comment('###############################################')
ctx.comment('Step '+str(STEP)+': '+STEPS[STEP]['description'])
ctx.comment('###############################################')
ctx.comment(' ')
i = 0
for s, d in zip(samples, pcr_wells_samples):
pick_up(m20)
move_vol_multichannel(m20, reagent = Samples, source = s, dest = d,
vol = VOLUME_SAMPLE, air_gap_vol = air_gap_sample, x_offset = x_offset,
pickup_height = 0.2, disp_height = -10, rinse = False,
blow_out=True, touch_tip=False)
m20.drop_tip(home_after = False)
tip_track['counts'][m20] += 8
i = i + 1
end = datetime.now()
time_taken = (end - start)
ctx.comment('Step ' + str(STEP) + ': ' +
STEPS[STEP]['description'] + ' took ' + str(time_taken))
STEPS[STEP]['Time:'] = str(time_taken)
# Export the time log to a tsv file
if not ctx.is_simulating():
with open(file_path, 'w') as f:
f.write('STEP\texecution\tdescription\twait_time\texecution_time\n')
for key in STEPS.keys():
row = str(key)
for key2 in STEPS[key].keys():
row += '\t' + format(STEPS[key][key2])
f.write(row + '\n')
f.close()
############################################################################
finish_run()
def run(ctx: protocol_api.ProtocolContext):
# load labware
ic_pk = ctx.load_labware(
'opentrons_96_aluminumblock_nest_wellplate_100ul', '9',
'chilled tubeblock for internal control and proteinase K (strip 1)'
).wells()[0]
bb = ctx.load_labware('nest_12_reservoir_15ml', '8',
'reagent reservoir Binding Buffer')
binding_buffer = bb.wells()[:2]
dest_plate = ctx.load_labware('nest_96_wellplate_2ml_deep', '11',
'96-deepwell sample plate')
# load tips
tips300 = [
ctx.load_labware('opentrons_96_filtertiprack_200ul', '5',
'200µl filter tiprack')
]
tips20 = [
ctx.load_labware('opentrons_96_filtertiprack_20ul', '6',
'20µl filter tiprack')
]
# load pipette
m300 = ctx.load_instrument('p300_multi_gen2', 'right', tip_racks=tips300)
s20 = ctx.load_instrument('p20_single_gen2', 'left', tip_racks=tips20)
m300.flow_rate.aspirate = 50
m300.flow_rate.dispense = 150
m300.flow_rate.blow_out = 300
s20.flow_rate.aspirate = 50
s20.flow_rate.dispense = 100
s20.flow_rate.blow_out = 300
# setup samples
num_cols = math.ceil(NUM_SAMPLES / 8)
bbs = bb.wells()[:2]
dests_single = dest_plate.wells()[:NUM_SAMPLES]
dests_multi = dest_plate.rows()[0][:num_cols]
tip_log = {'count': {}}
folder_path = '/data/A'
tip_file_path = folder_path + '/tip_log.json'
if TIP_TRACK and not ctx.is_simulating():
if os.path.isfile(tip_file_path):
with open(tip_file_path) as json_file:
data = json.load(json_file)
if 'tips1000' in data:
tip_log['count'][m300] = data['tips1000']
else:
tip_log['count'][m300] = 0
if 'tips20' in data:
tip_log['count'][s20] = data['tips20']
else:
tip_log['count'][s20] = 0
else:
tip_log['count'] = {m300: 0, s20: 0}
tip_log['tips'] = {
m300: [tip for rack in tips300 for tip in rack.wells()],
s20: [tip for rack in tips20 for tip in rack.rows()[0]]
}
tip_log['max'] = {pip: len(tip_log['tips'][pip]) for pip in [m300, s20]}
def pick_up(pip):
nonlocal tip_log
if tip_log['count'][pip] == tip_log['max'][pip]:
ctx.pause('Replace ' + str(pip.max_volume) +
'µl tipracks before \ resuming.')
pip.reset_tipracks()
tip_log['count'][pip] = 0
pip.pick_up_tip(tip_log['tips'][pip][tip_log['count'][pip]])
tip_log['count'][pip] += 1
heights = {tube: 20 for tube in binding_buffer}
# radius = (binding_buffer[0].diameter)/2
min_h = 5
def h_track(vol, tube):
nonlocal heights
dh = vol / (math.pi * (radius**2))
if heights[tube] - dh > min_h:
heights[tube] = heights[tube] - dh
else:
heights[tube] = min_h # stop 5mm short of the bottom
return heights[tube]
# transfer internal control + proteinase K
for d in dests_single:
pick_up(s20)
s20.transfer(ICPK_VOlUME,
ic_pk.bottom(2),
d.bottom(2),
air_gap=5,
new_tip='never')
s20.air_gap(5)
s20.drop_tip()
# transfer binding buffer
for i, e in enumerate(dests_multi):
pick_up(m300)
m300.transfer(BB_VOLUME,
bbs[i // 6].bottom(2),
e.bottom(10),
air_gap=5,
mix_after=(5, 100),
new_tip='never')
m300.air_gap(100)
m300.drop_tip()
ctx.comment('Terminado.')
# track final used tip
if not ctx.is_simulating():
if not os.path.isdir(folder_path):
os.mkdir(folder_path)
data = {'tips300': tip_log['count'][m300]}
with open(tip_file_path, 'w') as outfile:
json.dump(data, outfile)
def run(ctx: protocol_api.ProtocolContext):
[csv, m300_mount, p300_mount, temp_mod_temp, asp_rate_step1,
pbs_dispense_rate,
incubation_time, first_media_x, second_media_y, track_tips
] = get_values( # noqa: F821
"csv", "m300_mount", "p300_mount", "temp_mod_temp",
"asp_rate_step1", "pbs_dispense_rate",
"incubation_time", "first_media_x", "second_media_y", "track_tips")
# LABWARE
temp_mod = ctx.load_module('temperature module gen2', '10')
reagents = ctx.load_labware('nest_12_reservoir_15ml', '11')
waste_res = ctx.load_labware('nest_12_reservoir_15ml', '7')
plate = temp_mod.load_labware(
'corning_96_wellplate_360ul_flat', '10')
# TIPRACKS
tipracks = [ctx.load_labware('opentrons_96_filtertiprack_200ul', slot)
for slot in ['4', '5', '6']]
# INSTRUMENTS
p300 = ctx.load_instrument('p300_single_gen2',
p300_mount,
tip_racks=tipracks)
m300 = ctx.load_instrument('p300_multi_gen2',
m300_mount,
tip_racks=tipracks)
tips_by_col = [tip for rack in tipracks
for col in rack.columns() for tip in col[::-1]]
tip_cols = [tips_by_col[i:i+8] for i in range(0, len(tips_by_col), 8)]
""" TIP-TRACKING BETWEEN RUNS. """
total_tip_cols = 36
file_path = '/data/csv/tiptracking.json'
file_dir = os.path.dirname(file_path)
tips_by_col = [tip for rack in tipracks
for col in rack.columns() for tip in col[::-1]]
tip_cols = [tips_by_col[i:i+8] for i in range(0, len(tips_by_col), 8)]
if track_tips and not ctx.is_simulating():
# check for file directory
if not os.path.exists(file_dir):
os.makedirs(file_dir)
# if no file, then use standard tip_chunks definition, and the
# end of the code will write to updated tip_chunks list to
# the file created. This if statement handles the case in which
# tip tracking is selected for the first time.
if not os.path.isfile(file_path):
tip_chunks = [tips_by_col[i:i+8] for i in range(0,
len(tips_by_col), 8)]
else:
# grab nested list tip_chunks from file.
source = open(file_path, 'rb').read()
# see below for conversion of tip_chunks nested list to bools.
# in order to dump the well objects in tip_chunks to a json file,
# they had to be serializable (int, string, bool). The end of the
# protocol does this conversion.
tip_bool_chunks = json.loads(source)
# convert bools back to well objects to use.
tip_chunks = [[] for _ in range(total_tip_cols)]
for i, (bool_chunk, tip_chunk) in enumerate(zip(tip_bool_chunks,
tip_cols)):
if len(bool_chunk) == 0:
continue
for true_tip, tip_loc in zip(bool_chunk, tip_chunk):
if true_tip:
tip_chunks[i].append(tip_loc)
else:
continue
else:
# standard definition of tip_chunks if not tracking tips.
tip_chunks = [tips_by_col[i:i+8] for i in range(0,
len(tips_by_col), 8)]
"""PROTOCOL BEGINS """
csv_rows = [[val.strip() for val in line.split(',')]
for line in csv.splitlines()
if line.split(',')[0].strip()][1:]
"""FIND INVOLVED WELLS"""
values_from_csv = []
wells_from_csv = []
for row in csv_rows:
well, value = row[:2]
value = int(value)
values_from_csv.append(value)
wells_from_csv.append(well)
# create nested list of all values in csv (by column).
value_chunk_cols = [values_from_csv[i:i+8]
for i in range(0, len(values_from_csv), 8)]
list_well_tips = []
"""CREATE A LIST OF # TIPS FOR EACH WELL"""
start_point = 0
tip_count = 0
for i, chunk in enumerate(value_chunk_cols):
start_point = 0
# check for the values in each column.
# if we find a well with a value of 85 or greater,
# use that index (j) as the starting point and see how many values
# after that are also greater than 85. Once we don't find one,
# break.
for j, value in enumerate(chunk[start_point:]):
if value >= 85:
for check_values in chunk[j:]:
if check_values >= 85:
tip_count += 1
else:
break
list_well_tips.append(tip_count)
tip_count = 0
continue
else:
list_well_tips.append(0)
# create a dictionary which says how many tips go to each well.
# For example, if the entire first column has values higher than 85,
# the first value in the dictionary will be "A1: 8". If first four wells
# in column 2 are greater than 85, "B1:4".
dict_tips_per_well = {}
tip_ctr = 0
for j, (well, num_tips) in enumerate(zip(wells_from_csv,
list_well_tips)):
if tip_ctr > 0:
tip_ctr -= 1
continue
if num_tips > 0:
tip_ctr = num_tips - 1
dict_tips_per_well[well] = num_tips
# print('\n\n', dict_tips_per_well, '\n\n')
"""PICKUP FUNCTION"""
def pick_up(num_channels_per_pickup):
nonlocal tip_chunks
if num_channels_per_pickup > 1:
pip = m300
else:
pip = p300
try:
col = 0
# based on the demand of the next well (1-8 tips), this for loop
# will find the first available column having adequate number
# of tips starting from the first. If the tip pick up order is:
# 6, 8, 2 for the first 3 pick ups, then 6 tips will be taken from
# column 1, 8 tips from column 2, and 2 tips back from column 1.
# efficient tip pick up instead of "throwing away" a whole column
# after pick up.
for _ in range(36):
if num_channels_per_pickup <= len(tip_chunks[col]):
break
else:
col += 1
pip.pick_up_tip(tip_chunks[col][num_channels_per_pickup-1])
# remove as many tips as we picked up in that column
# from the 0 index.
for _ in range(num_channels_per_pickup):
tip_chunks[col].pop(0)
# replace tip exception
except IndexError:
ctx.pause("Replace empty tip racks on slots 4, 5, and 6")
pip.reset_tipracks()
tip_chunks = [tips_by_col[i:i+8] for i in range(0,
len(tips_by_col), 8)]
col = 0
for _ in range(36):
if num_channels_per_pickup <= len(tip_chunks[col]):
break
else:
col += 1
pip.pick_up_tip(tip_chunks[col][num_channels_per_pickup-1])
for _ in range(num_channels_per_pickup):
tip_chunks[col].pop(0)
if len(tip_chunks[col]) == 0:
tip_chunks.remove(tip_chunks[col])
# DUMP WASTE
vol_ctr = 0
waste_well = 0
# move to next well in reservoir once we fill one.
def check_waste_vol(vol):
nonlocal vol_ctr
nonlocal waste_well
vol_ctr += vol
if vol_ctr > 12000:
waste_well += 1
vol_ctr = 0
waste = waste_res.wells()[waste_well]
temp_mod.set_temperature(temp_mod_temp)
ctx.pause("""
Ensure temperature module is at correct temperature, then,
select "Resume" on the Opentrons app.
""")
# REAGENTS
pbs = reagents.wells()[0]
trypsin = reagents.wells()[1]
media = reagents.wells()[-1]
airgap = 20
ctx.comment("MOVING INCLUDED WELLS TO WASTE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
# aspirate from side so as to not disturb cell culture.
pip.aspirate(200, plate_well.bottom(z=1).move(
Point(x=(plate_well.diameter/2-2))), rate=asp_rate_step1)
pip.dispense(200, waste)
check_waste_vol(200)
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
ctx.comment("\n\n\nMOVING PBS TO PLATE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
pip.aspirate(150, pbs, rate=pbs_dispense_rate)
pip.dispense(150, plate_well.bottom(z=1).move(
Point(x=(plate_well.diameter/2-2))))
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
ctx.comment("\n\n\nREMOVING PBS FROM PLATE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
pip.aspirate(175, plate_well.bottom(z=1).move(
Point(x=(plate_well.diameter/2-2))))
pip.dispense(175, waste)
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
ctx.comment("\n\n\nMOVING TRYPSIN TO PLATE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
pip.aspirate(25, trypsin)
pip.dispense(25, plate_well)
pip.blow_out()
pip.touch_tip()
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
ctx.delay(minutes=incubation_time)
ctx.comment("\n\n\nMOVING MEDIA TO PLATE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
pip.aspirate(140, media)
pip.dispense(140, plate_well)
pip.blow_out()
pip.touch_tip()
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
ctx.comment("\n\n\nASPIRATE FIRST MEDIA FROM PLATE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
pip.aspirate(first_media_x, plate_well.bottom(z=1).move(
Point(x=(plate_well.diameter/2-2))))
pip.dispense(first_media_x, waste)
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
ctx.comment("\n\n\nDISPENSE SECOND MEDIA TO PLATE")
for i, well in enumerate(dict_tips_per_well):
num_tips = dict_tips_per_well[well]
plate_well = plate.wells_by_name()[well]
if num_tips > 1:
pip = m300
else:
pip = p300
pick_up(num_tips)
pip.aspirate(second_media_y, media)
pip.dispense(second_media_y, plate_well)
pip.air_gap(airgap)
pip.drop_tip()
ctx.comment('\n')
tip_data = []
for i, chunk in enumerate(tip_chunks):
tip_data.append([])
if len(chunk) > 0:
for value in chunk:
tip_data[i].append(True)
else:
continue
# write to the ot-2 no matter what in case the user would like to start
# tracking tips for the next run
if not ctx.is_simulating():
with open(file_path, 'w') as outfile:
outfile.write(json.dumps(tip_data))