def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix;
Adding samples from different labware.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
# Pipette tips
if dispension_vol >= 19:
## When the mm volume to be dispensed >= 19, 200µL tips are
## needed in addition to the 20µL tips.
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
4,
'200tips')
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
7,
'20tips_1')
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
10,
'20tips_2')
tips_20 = [tips_20_1, tips_20_2]
else:
## When the mm volume to be dispensed <=19, only 20µL are needed
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
4,
'20tips_1')
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
7,
'20tips_2')
tips_20_3 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
10,
'20tips_3')
tips_20 = [tips_20_1, tips_20_2, tips_20_3]
# Tube_racks & plates
destination_plate = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
3,
'plate_96')
if mastermix_tube_type == 'tube_1.5mL':
mastermix_tube = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
1,
'mastermix_tube')
if sample_tube_type == 'tube_1.5mL':
sample_source_1 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
2,
'sample_source_1')
sample_source_2 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
5,
'sample_source_2')
sample_source_3 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
8,
'sample_source_3')
sample_source_4 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
11,
'sample_source_4')
if sample_tube_type == 'plate_96':
sample_source_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
2,
'sample_source_1')
sample_source_2 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
5,
'sample_source_2')
if simulate: #Simulator
if mastermix_tube_type == 'tube_5mL':
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
mastermix_tube = protocol.load_labware_from_definition(
labware_def_5mL,
1,
'mastermix_tube')
if sample_tube_type == 'PCR_strip':
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
sample_source_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
2,
'sample_source_1')
sample_source_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
5,
'sample_source_2')
sample_source_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
8,
'sample_source_3')
sample_source_4 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
11,
'sample_source_4')
else:
if mastermix_tube_type == 'tube_5mL':
mastermix_tube = protocol.load_labware(
'eppendorfscrewcap_15_tuberack_5000ul',
1,
'mastermix_tube')
if sample_tube_type == 'PCR_strips':
sample_source_1 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul',
2,
'sample_source_1')
sample_source_2 = protocol.load_labware_(
'pcrstrips_96_wellplate_200ul',
5,
'sample_source_2')
sample_source_3 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul',
8,
'sample_source_3')
sample_source_4 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul',
11,
'sample_source_4')
# Pipettes
if dispension_vol >= 19:
p300 = protocol.load_instrument(
'p300_single_gen2',
'right',
tip_racks=[tips_200])
p20 = protocol.load_instrument(
'p20_single_gen2',
'left',
tip_racks=tips_20)
# =============================================================================
# PREDIFINED VARIABLES=========================================================
# =============================================================================
aspiration_vol = dispension_vol + (dispension_vol/100*2)
## The aspiration_vol is the volume (µL) that is aspirated from the
## container.
##### Variables for volume tracking
start_height = vt.cal_start_height(mastermix_tube_type, start_vol)
## Call start height calculation function from volume tracking module.
current_height = start_height
## Set the current height to start height at the beginning of the
## protocol.
sample_mix_vol = sample_vol + 3
## primer_mix_vol = volume for pipetting up and down
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
# Setting starting tip
if dispension_vol >= 19:
## If the mm volume to be dispendsed >= 19, assign p300 starting tip
p300.starting_tip = tips_200.well(starting_tip_p200)
p20.starting_tip = tips_20_1.well(starting_tip_p20)
# Mastermix tube location
MasterMix = mastermix_tube[mastermix_source]
# Make a list of all possible wells in the destination plate
destination_wells = []
for well in destination_plate.wells():
destination_wells.append(well)
# Create a list of wells where samples should go
sample_wells = destination_wells[:number_of_samples*replicates]
#Create a list of wells where standard sample replicates should go
slice_std_sample_wells = slice(
(number_of_samples * replicates), (number_of_samples * replicates)+ number_of_std_samples)
std_sample_wells = destination_wells[slice_std_sample_wells]
## Slice the list after the number of samples, and after the number
## of standard sample replicates to retrieve the wells in between
# Create the list of wells where NTCs should go
slice_NTC_wells = slice(
(number_of_samples * replicates)+ number_of_std_samples,
(number_of_samples * replicates)+ number_of_std_samples + number_of_NTCs)
NTC_wells = destination_wells[slice_NTC_wells]
## Slice the list after the number of samples + the number of standard
## sample replicates, and after that + the number of NTCs to retrieve
## the wells in between (the last of the wells that need to be filled)
# Create the list of wells where standerd series should go
std_series_wells = []
std_series_columns = (
[destination_plate.columns_by_name()[column_name] for column_name in
['12', '11', '10']])
std_series_columns = std_series_columns[:number_of_std_series]
## Reserve a column at the end of the plate for every std_series
## Separate the columns into wells and append them to list
for column in std_series_columns:
column = column[:length_std_series]
## cut off the columns after a certain std_series length
for well in column:
std_series_wells.append(well)
# Create a list with wells where mastermix should go
MasterMixAliquots = (
sample_wells + std_sample_wells + NTC_wells + std_series_wells)
if redo:
## This is the part of the protocol that accesses the specific
## sample wells specified in the variables to set
## NOTE: this only works for 96wells plate, when you need to redo
## samples with other labware you can rearrange the order of tubes
sample_sources = []
wells_sample_source_1 = (
[sample_source_1.wells_by_name()[well_name] for well_name in
samples_sample_source_1])
for well in wells_sample_source_1:
sample_sources.append(well)
wells_sample_source_2 = (
[sample_source_2.wells_by_name()[well_name] for well_name in
samples_sample_source_2])
for well in wells_sample_source_2:
sample_sources.append(well)
wells_sample_source_3 = (
[sample_source_3.wells_by_name()[well_name] for well_name in
samples_sample_source_3])
for well in wells_sample_source_3:
sample_sources.append(well)
wells_sample_source_4 = (
[sample_source_4.wells_by_name()[well_name] for well_name in
samples_sample_source_4])
for well in wells_sample_source_4:
sample_sources.append(well)
else:
## This is the part of the protocol that access the sample wells in
## a normal PCR (where you don't need to access specific wells)
sample_source_wells = []
sample_source_wells_string = []
## The string list is needed to be able to start at another well
if sample_tube_type == 'tube_1.5mL' or sample_tube_type == 'plate_96':
if sample_racks >= 1:
for well in sample_source_1.wells():
sample_source_wells.append(well)
sample_source_wells_string.append(str(well))
if sample_racks >= 2:
for well in sample_source_2.wells():
sample_source_wells.append(well)
sample_source_wells_string.append(str(well))
if sample_racks >= 3:
for well in sample_source_3.wells():
sample_source_wells.append(well)
sample_source_wells_string.append(str(well))
if sample_racks >= 4:
for well in sample_source_4.wells():
sample_source_wells.append(well)
sample_source_wells_string.append(str(well))
if sample_tube_type == 'PCR_strip':
sample_source_columns = (
([sample_source_1.columns_by_name()[column_name]
for column_name in sample_columns]))
if sample_racks >= 2:
sample_columns_2 = (
([sample_source_2.columns_by_name()[column_name]
for column_name in sample_columns]))
for column in sample_columns_2:
sample_source_columns.append(column)
if sample_racks >= 3:
sample_columns_3 = (
([sample_source_3.columns_by_name()[column_name]
for column_name in sample_columns]))
for column in sample_columns_3:
sample_source_columns.append(column)
if sample_racks >= 4:
sample_columns_4 = (
([sample_source_4.columns_by_name()[column_name]
for column_name in sample_columns]))
for column in sample_columns_4:
sample_source_columns.append(column)
## Make a list of columns, this is a list of lists!
for column in sample_source_columns:
for well in column:
sample_source_wells.append(well)
sample_source_wells_string.append(str(well))
## Separate the columns into wells and append them to list
## Cut slice out off list of sample_sources, starting with the
## indicated first sample and ending after the number_of_samples
first_sample_index = sample_source_wells_string.index(
first_sample + ' of sample_source_1 on 2')
## Determine the index of the first sample in the list made from
## strings -- we cannot find strings in the normal robot list
## so we needed to convert the wells to strings.
slice_sample_sources = slice(
first_sample_index,
first_sample_index + number_of_samples)
## Determine the slice
## Determine position of the std_sample
slice_std_sample = slice(
first_sample_index + number_of_samples,
first_sample_index + number_of_samples + 1)
## If you have a std sample included, this will be added as the last sample
std_source = sample_source_wells[slice_std_sample] * number_of_std_samples
## Makes a list of len number_of_std_samples of the std_source well
## Cut sample slice out of sample_source_wells list
sample_sources = sample_source_wells[slice_sample_sources]
## If replicates are desired, this copies the sample_sources list
sample_sources = sample_sources * replicates
## If you have a std sample included, this will add the std_samples
## to the end of the sample_sources list
sample_sources = sample_sources + std_source
# =============================================================================
## PIPETTING===================================================================
## ============================================================================
## LIGHTS----------------------------------------------------------------------
if qPCR:
protocol.set_rail_lights(False)
if not qPCR:
protocol.set_rail_lights(True)
## ----------------------------------------------------------------------------
## ALIQUOTING MASTERMIX--------------------------------------------------------
if dispension_vol >= 19:
pipette = p300
else:
pipette = p20
for i, well in enumerate(MasterMixAliquots):
## aliquot mix, for each well do the following:
if i == 0:
pipette.pick_up_tip()
## If we are at the first well, start by picking up a tip.
elif i % 8 == 0:
pipette.drop_tip()
pipette.pick_up_tip()
## Then, after every 8th well, drop tip and pick up new
current_height, pip_height, bottom_reached = vt.volume_tracking(
mastermix_tube_type, dispension_vol, current_height)
## call volume_tracking function, obtain current_height,
## pip_height and whether bottom_reached.
if bottom_reached:
aspiration_location = MasterMix.bottom(z=1)
protocol.comment("You've reached the bottom of the tube!")
## If bottom is reached keep pipetting from bottom + 1
else:
aspiration_location = MasterMix.bottom(pip_height)
## Set the location of where to aspirate from.
#### The actual aliquoting of mastermix
pipette.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the
## location specified in aspiration_location.
pipette.dispense(dispension_vol, well)
## Dispense the amount specified in dispension_vol to the
## location specified in well (so a new well every time the
## loop restarts)
pipette.dispense(10, aspiration_location)
## Alternative for blow-out, make sure the tip doesn't fill
## completely when using a disposal volume by dispensing some
## of the volume after each pipetting step. (blow-out to many
## bubbles)
pipette.drop_tip()
## ----------------------------------------------------------------------------
## ADDING SAMPLES--------------------------------------------------------------
## Loop through source and destination wells
for sample_tube, well in zip(sample_sources, destination_plate.wells()):
p20.pick_up_tip()
p20.aspirate(sample_vol, sample_tube)
p20.dispense(sample_vol, well)
sample_mix_vol = sample_vol + 3
## primer_mix_vol = volume for pipetting up and down
p20.mix(3, sample_mix_vol, well)
p20.dispense(10, well)
p20.drop_tip()
# ----------------------------------------------------------------------------
## LIGHTS----------------------------------------------------------------------
if not qPCR:
protocol.set_rail_lights(False)
# ----------------------------------------------------------------------------
# =============================================================================
