def run(protocol: protocol_api.ProtocolContext):
"""
During the qPCR (project_related/Dina/21june_qPCRs_DINA_EVE.py) we take
sample, dilute it 100x (pipetting up-and-down 3x with 4.5uL) and then
directly transfer the dilution to the PCR plate. With the sample_mix, which
was added to the plate 8x I saw a lot of variation in Cq values. I'm under
the impression mixing was not sufficient. In this protocol I want to
compare some different mixing techniques, to see if that can be improved.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
##### Loading labware
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
3, #deck position
'tips_200') #custom name
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
10, #deck position
'tips_20') #custom name
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
7, #deck position
'tips_20') #custom name
plate_96_qPCR = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
5, #deck position
'plate_96_qPCR') #custom name
# ##### !!! FOR ROBOT
# sample_strips_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 1, #deck position
# 'sample_strips_1') #custom name
# dilution_strips = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 4, #deck position
# 'dilution_strips') #custom name
# tubes_5mL = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware definition
# 6, #deck position
# 'tubes_5mL') #custom name
#### !!! FOR SIMULATOR
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
sample_strips_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
1,
'sample_strips_1')
dilution_strips = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
4,
'dilution_strips')
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
tubes_5mL = protocol.load_labware_from_definition(
labware_def_5mL, #variable derived from opening json
6,
'5mL_tubes')
##### Loading pipettes
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
p20 = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=[tips_20_1, tips_20_2]) #assigned tiprack
# =============================================================================
# VARIABLES TO SET#!!!=========================================================
# =============================================================================
start_vol_mix = 2178
## The start_vol_m is the volume (ul) of mix that is in the source ##
## labware at the start of the protocol. ##
start_vol_water = 5000
## The start_vol_w is the volume (ul) of water that is in the source ##
## labware at the start of the protocol. ##
##!!! Fill up to above 5mL line ##
dispension_vol_mix = 22
## The dispension_vol_m is the volume (ul) of mastermix that needs to ##
## be aliquoted into the destination wells/tubes. ##
dispension_vol_water = 148.5
## The dil_vol_w is the volume of water to be pipetted for the ##
## dilution. ##
sample_vol_dil = 1.5
## The dil_vol_s is the volume of sample to be pipetted for the ##
## 100x dilution. ##
sample_vol_pcr = 3
## The sample_vol is the volume (ul) of sample added to the PCR ##
## reaction. ##
p300.starting_tip = tips_200.well('A1')
p20.starting_tip = tips_20_1.well('A1')
## The starting_tip is the location of first pipette tip in the box ##
#### Which wells/tubes are used
mastermix = tubes_5mL['C1']
H2O = ([
tubes_5mL.wells_by_name()[well_name]
for well_name in ['B1', 'B2', 'B3']
])
sample = sample_strips_1['A1']
## where to put mastermix
PCR_plate = []
PCR_plate_columns = ([
plate_96_qPCR.columns_by_name()[column_name] for column_name in
['1', '2', '3', '5', '6', '7', '8', '9', '10', '11']
])
for column in PCR_plate_columns:
for well in column:
PCR_plate.append(well)
for well in ([
plate_96_qPCR.wells_by_name()[well_name]
for well_name in ['G12', 'H12']
]):
PCR_plate.append(well)
## where to put water for dilutions
dilution_plate = dilution_strips.columns_by_name()['1']
# =============================================================================
# PREDIFINED VARIABLES=========================================================
# =============================================================================
container_mix = container_water = 'tube_5mL'
##### Variables for volume tracking
start_height_mix = vt.cal_start_height(container_mix, start_vol_mix)
start_height_water = vt.cal_start_height(container_water, start_vol_water)
# =============================================================================
# ALIQUOTING DILUTION WATER AND MASTERMIX======================================
# =============================================================================
aliquots = ['water', 'PCR_mix']
## what will be aliquoted in this protocol
for aliquot in aliquots:
if aliquot == 'PCR_mix':
source = mastermix
destination = PCR_plate
current_height = start_height_mix
container = container_mix
dispension_vol = dispension_vol_mix
elif aliquot == 'water':
counter = 0 # how many tubes emptied
source = H2O[counter]
destination = dilution_plate
current_height = start_height_water
container = container_water
dispension_vol = dispension_vol_water
for i, well in enumerate(destination):
## aliquot mix in entire qPCR plate, for each well do the following:
aspiration_vol = dispension_vol + (dispension_vol / 100 * 2)
## Set correct variables for volume_tracking
if i == 0:
p300.pick_up_tip()
## If we are at the first well, start by picking up a tip.##
elif i % 8 == 0:
p300.drop_tip()
p300.pick_up_tip()
## Then, after every 8th well, drop tip and pick up new ##
current_height, pip_height, bottom_reached = vt.volume_tracking(
container, dispension_vol, current_height)
## call volume_tracking function, obtain current_height, ##
## pip_height and whether bottom_reached. ##
if bottom_reached:
if aliquot == 'water':
## continue with next tube, reset vt
current_height = start_height_water
current_height, pip_height, bottom_reached = (
vt.volume_tracking(container, dispension_vol,
current_height))
counter = counter + 1
source = H2O[counter]
aspiration_location = source.bottom(current_height)
protocol.comment("Continue with next tube of water")
elif aliquot == 'PCR_mix':
aspiration_location = source.bottom(z=1)
protocol.comment("You've reached the bottom of the tube!")
else:
aspiration_location = source.bottom(pip_height)
## Set the location of where to aspirate from. ##
#### The actual aliquoting of mastermix
p300.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
p300.dispense(dispension_vol, well)
## Dispense the amount specified in dispension_vol to the ##
## location specified in well (looping through plate) ##
p300.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 too many##
## bubbles) ##
p300.drop_tip()
## when entire plate is full, drop tip ##
# =============================================================================
# DILUTING AND DISTRIBUTING SAMPLE MIX=========================================
# =============================================================================
for mix_method in range(8):
## where to dilute
dilution = dilution_plate[mix_method]
## set variables per mix_method
if mix_method == 0:
## 1: Mixing 3x with sample_vol +3. Distribute in 24 wells
mix_vol = sample_vol_dil + 3
number_of_mixes = 3
destination = PCR_plate[:24]
elif mix_method == 1:
## 2: Mixing 5x with sample_vol + 3
mix_vol = sample_vol_dil + 3
number_of_mixes = 5
destination = PCR_plate[24:32]
elif mix_method == 2:
## 3: Mixing 7x with sample_vol + 3
mix_vol = sample_vol_dil + 3
number_of_mixes = 7
destination = PCR_plate[32:40]
elif mix_method == 3:
## 4: Mixing 10x with sample_vol + 3
mix_vol = sample_vol_dil + 3
number_of_mixes = 10
destination = PCR_plate[40:48]
elif mix_method == 4:
## 5: Mixing 3x with 20µL
mix_vol = 20
number_of_mixes = 3
destination = PCR_plate[48:56]
elif mix_method == 5:
## 6: Mixing 5x with 20µL
mix_vol = 20
number_of_mixes = 5
destination = PCR_plate[56:64]
elif mix_method == 6:
## 7: Mixing 7x with 20µL
mix_vol = 20
number_of_mixes = 7
destination = PCR_plate[64:72]
elif mix_method == 7:
## 8: Mixing 10x with 20µL
mix_vol = 20
number_of_mixes = 10
destination = PCR_plate[72:80]
#### diluting sample ##
p20.pick_up_tip()
## p20 picks up tip from location of specified starting_tip or ##
## the following. ##
p20.aspirate(sample_vol_dil, sample)
## aspirate sample_volume_dil = volume for dil. from sample_mix ##
p20.dispense(sample_vol_dil, dilution)
## dispense in dilution tube
p20.mix(number_of_mixes, mix_vol, dilution)
## mix according to mix_method
p20.dispense(10, dilution)
## instead of blow-out ##
p20.drop_tip()
## Drop tip in trashbin on 12. ##
#### Distribute from dilution to PCR plate ##
for well in destination:
p20.pick_up_tip()
p20.aspirate(sample_vol_pcr, dilution)
## aspirate sample_vol_pcr from dilution ##
p20.dispense(sample_vol_pcr, well)
## dispense into pcr_well ##
p20.mix(3, 20, well)
## Mix 3 times up and down with max pipette vol ##
p20.dispense(10, well)
## instead of blow-out. ##
p20.drop_tip()
def run(protocol: protocol_api.ProtocolContext):
"""
Pick up 200µL filter tip. Aspirate 200µL from 50mL tube and transfer
5x to 1x 1.5mL tube (1mL aliquots) - repeat for 24x 1.5mL tubes.
Drop tip, pick up new tip and repeat.
1x 50mL tube for 48x 1.5mL tubes.
"""
# =============================================================================
# =====================LOADING LABWARE AND PIPETTES============================
# =============================================================================
# Pipette tips
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware def
10, #deck position
'200tips') #custom name
# Tube racks
stock_tubes = protocol.load_labware(
'opentrons_6_tuberack_falcon_50ml_conical', #labware def
7, #deck position
'50mL_tubes') #custom name
aliquot_tubes_1 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
4, #deck position
'aliquot_tubes_1') #custom name
aliquot_tubes_2 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
1, #deck position
'aliquot_tubes_2') #custom name
aliquot_tubes_3 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
11, #deck position
'aliquot_tubes_3') #custom name
aliquot_tubes_4 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
8, #deck position
'aliquot_tubes_4') #custom name
aliquot_tubes_5 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
5, #deck position
'aliquot_tubes_5') #custom name
aliquot_tubes_6 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
2, #deck position
'aliquot_tubes_6') #custom name
aliquot_tubes_7 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
6, #deck position
'aliquot_tubes_7') #custom name
aliquot_tubes_8 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
3, #deck position
'aliquot_tubes_8') #custom name
# Pipettes
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
# =============================================================================
# ==========================VARIABLES TO SET#!!!===============================
# =============================================================================
start_vol = 50000
## The start_vol is the volume (ul) that is in the source labware at ##
## the start of the protocol. ##
dispension_vol = 200
## The dispension_vol is the volume (ul) that needs to be aliquoted ##
## into the destination wells/tubes. ##
p300.starting_tip = tips_200.well('E6')
## The starting_tip is the location of first pipette tip in the box ##
container = 'tube_50mL'
## The container variable is needed for the volume tracking module. ##
## It tells the module which dimensions to use for the calculations ##
## of the pipette height. It is the source labware from which liquid ##
## is aliquoted. ##
## There are several options to choose from: ##
## 'tube_1.5ml', 'tube_2mL', 'tube_5mL', 'tube_15mL', 'tube_50mL' ##
number_of_source_wells = 3
# source and destination wells=================================================
source_wells = []
destination_wells = []
if number_of_source_wells == 1:
source_well_1 = stock_tubes['A1']
source_wells.append(
source_well_1)
destinations = (
aliquot_tubes_1.wells() + aliquot_tubes_2.wells())
for well in destinations:
destination_wells.append(well)
elif number_of_source_wells == 2:
source_well_1 = stock_tubes['A1']
source_well_2 = stock_tubes['A2']
sources = (source_well_1, source_well_2)
source_wells.append(sources)
destinations = (
aliquot_tubes_1.wells() + aliquot_tubes_2.wells() +
aliquot_tubes_3.wells() + aliquot_tubes_4.wells())
for well in destinations:
destination_wells.append(well)
elif number_of_source_wells == 3:
source_well_1 = stock_tubes['A1']
source_well_2 = stock_tubes['A2']
source_well_3 = stock_tubes['B1']
sources = (source_well_1, source_well_2, source_well_3)
source_wells.append(sources)
destinations = (
aliquot_tubes_1.wells() + aliquot_tubes_2.wells() +
aliquot_tubes_3.wells() + aliquot_tubes_4.wells() +
aliquot_tubes_5.wells() + aliquot_tubes_6.wells())
for well in destinations:
destination_wells.append(well)
elif number_of_source_wells == 4:
source_well_1 = stock_tubes['A1']
source_well_2 = stock_tubes['A2']
source_well_3 = stock_tubes['B1']
source_well_4 = stock_tubes['B2']
sources = (source_well_1, source_well_2, source_well_3, source_well_4)
source_wells.append(sources)
destinations = (
aliquot_tubes_1.wells() + aliquot_tubes_2.wells() +
aliquot_tubes_3.wells() + aliquot_tubes_4.wells() +
aliquot_tubes_5.wells() + aliquot_tubes_6.wells() +
aliquot_tubes_7.wells() + aliquot_tubes_8.wells())
for well in destinations:
destination_wells.append(well)
# =============================================================================
# ==========================PREDIFINED VARIABLES===============================
# =============================================================================
aspiration_vol = dispension_vol
## The aspiration_vol is the volume (ul) that is aspirated from the ##
## container. ##
##### Variables for volume tracking
start_height = vt.cal_start_height(container, 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. ##
# =============================================================================
# =================================ALIQUOTING==================================
# =============================================================================
## For each column in destination_wells, pick up a tip, than for each ##
## well in these columns pipette mix, and after the+ column drop the tip##
## Repeat untill all columns in the list are done. ##
for i, well in enumerate(destination_wells):
## Name all the wells in the plate 'well', for all these do: ##
if i == 0:
p300.pick_up_tip()
## If we are at the first well, start by picking up a tip. ##
elif i % 24 == 0:
p300.drop_tip()
p300.pick_up_tip()
## Then, after every 8th well, drop tip and pick up a new one. ##
current_height, pip_height, bottom_reached = vt.volume_tracking(
container, dispension_vol, current_height)
## The volume_tracking function needs the arguments container, ##
## dispension_vol, and the current_height which we have set in ##
## this protocol. With those variables, the function updates ##
## the current_height, the pip_height and calculates the ##
## delta_height of the liquid after the next aspiration step. ##
if bottom_reached:
aspiration_location = source_wells.bottom(z=1) #!!!
protocol.comment("You've reached the bottom!")
else:
aspiration_location = source_wells.bottom(pip_height) #!!!
## If the level of the liquid in the next run of the loop will ##
## be smaller than 1 we have reached the bottom of the tube. ##
## To prevent the pipette from crashing into the bottom, we ##
## tell it to go home and pause the protocol so that this can ##
## never happen. Set the location of where to aspirate from. ##
## Because we put this in the loop, the location will change ##
## to the newly calculated height after each pipetting step. ##
p300.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
p300.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) ##
p300.drop_tip()
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix from a 1.5mL or 5mL tube.
Adding barcoded primers - from 1.5mL tubes, 2 different R primers.
"""
# =============================================================================
# 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', 2,
'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', 2,
'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
F_primer_source = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', 11,
'F_primer_source')
Ra_primer_source = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', 8,
'Ra_primer_source')
Rb_primer_source = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', 5,
'Rb_primer_source')
if mastermix_tube_type == 'tube_1.5mL':
mastermix_tube = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', 3,
'mastermix_tube')
if simulate: #Simulator
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
PCR_strips = protocol.load_labware_from_definition(
labware_def_pcrstrips, 6, 'PCR_strips')
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, 3, 'mastermix_tube')
else: #Robot
PCR_strips = protocol.load_labware('pcrstrips_96_wellplate_200ul', 6,
'PCR_strips')
if mastermix_tube_type == 'tube_5mL':
mastermix_tube = protocol.load_labware(
'eppendorfscrewcap_15_tuberack_5000ul', 3, 'mastermix_tube')
# 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.
F_primer_mix_vol = F_primer_vol + 3
R_primer_mix_vol = R_primer_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]
# Create a list of wells where mix and primers should go
destination_wells = []
PCR_strips_columns = ([
PCR_strips.columns_by_name()[column_name]
for column_name in ['2', '7', '11']
])
for column in PCR_strips_columns:
for destination_well in column:
destination_wells.append(destination_well)
MasterMixAliquots = destination_wells[:number_of_primers]
## cuts off the list after a the number_of_primers number of wells
F_primer_source_wells = F_primer_source.wells()[:number_of_primers]
Ra_primer_source_wells = Ra_primer_source.wells()[:number_of_primers]
Rb_primer_source_wells = Rb_primer_source.wells()[:number_of_primers]
# =============================================================================
## PIPETTING===================================================================
## ============================================================================
## LIGHTS----------------------------------------------------------------------
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 F PRIMERS TO THE MIX-------------------------------------------------
for F_primer_well, destination_well in zip(F_primer_source_wells,
MasterMixAliquots):
## Loop trough primer_wells and sample_wells
p20.pick_up_tip()
p20.aspirate(F_primer_vol, F_primer_well)
p20.dispense(F_primer_vol, destination_well)
p20.mix(3, F_primer_mix_vol, destination_well)
p20.dispense(10, destination_well)
p20.drop_tip()
# ## ----------------------------------------------------------------------------
# ## ADDING Ra PRIMERS TO THE MIX------------------------------------------------
for Ra_primer_well, destination_well in zip(Ra_primer_source_wells,
MasterMixAliquots):
## Loop trough primer_wells and sample_wells
p20.pick_up_tip()
p20.aspirate(R_primer_vol, Ra_primer_well)
p20.dispense(R_primer_vol, destination_well)
p20.mix(3, R_primer_mix_vol, destination_well)
p20.dispense(10, destination_well)
p20.drop_tip()
# ## ----------------------------------------------------------------------------
# ## ADDING Rb PRIMERS TO THE MIX-------------------------------------------------
for Rb_primer_well, destination_well in zip(Rb_primer_source_wells,
MasterMixAliquots):
## Loop trough primer_wells and sample_wells
p20.pick_up_tip()
p20.aspirate(R_primer_vol, Rb_primer_well)
p20.dispense(R_primer_vol, destination_well)
p20.mix(3, R_primer_mix_vol, destination_well)
p20.dispense(10, destination_well)
p20.drop_tip()
## ----------------------------------------------------------------------------
## LIGHTS----------------------------------------------------------------------
protocol.set_rail_lights(False)
# ----------------------------------------------------------------------------
## ============================================================================
def run(protocol: protocol_api.ProtocolContext):
"""
General protocol for multiple qPCRs of a batch.
Define if and how many dilution series you want to include. Usually only
in the first PCR of a batch, 3 dilution series are included.
Aliquoting Phusion PCRmix with EvaGreen added from a 5 mL tube to a
96-wells plate; use volume tracking.
After that, add samples to PCR mix.
Also include a standard sample mix (distribute to an entire column).
Dilution series should be added by hand in the first (how many you chose)
columns.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
##### Loading labware
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
3, #deck position
'tips_200') #custom name
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
10, #deck position
'tips_20') #custom name
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
7, #deck position
'tips_20') #custom name
plate_96_qPCR = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
5, #deck position
'plate_96_qPCR') #custom name
plate_96_dil_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
4, #deck position
'plate_96_dil') #custom name
plate_96_dil_2 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
1, #deck position
'plate_96_dil_2') #custom name
##### !!! FOR ROBOT
# tubes_5mL = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware definition
# 6, #deck position
# 'tubes_5mL') #custom name
# #### !!! FOR SIMULATOR
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
tubes_5mL = protocol.load_labware_from_definition(
labware_def_5mL, #variable derived from opening json
6,
'5mL_tubes')
##### Loading pipettes
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
p20 = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=[tips_20_1, tips_20_2]) #assigned tiprack
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
##### Setting starting tip ##
p300.starting_tip = tips_200.well(starting_tip_p200)
p20.starting_tip = tips_20_1.well(starting_tip_p20)
## The starting_tip is the location of first pipette tip in the box ##
##### Tube locations ##
MasterMix = tubes_5mL['C1']
## Location of the 5mL tube with mastermix ##
#### Where should mastermix go ##
number_of_wells = (number_of_samples + (number_std_series * 8) +
number_of_samplemix + number_of_NTCs)
##How many wells do need to be filled with mastermix ##
MasterMixAliquots = []
for well in plate_96_qPCR.wells():
MasterMixAliquots.append(well)
## Make a list with all wells of the plate ##
MasterMixAliquots = MasterMixAliquots[:number_of_wells]
## cuts off the list after a certain number of samples ##
#### Where are the samples located ##
samples = []
samples_string = []
for well in plate_96_dil_1.wells():
samples.append(well)
samples_string.append(str(well))
for well in plate_96_dil_2.wells():
samples.append(well)
## Makes a list of all wells in 2 full plates ##
index_first_sample = samples_string.index(first_sample +
' of plate_96_dil on 4')
samples = samples[index_first_sample:]
## starts sample location at firts sample ##
samples = samples[:number_of_samples]
## Cuts off the list after certain number of samples ##
#### Where do the samples go in the PCR plate ##
sample_dest = []
for well in plate_96_qPCR.wells():
sample_dest.append(well)
sample_dest = sample_dest[((number_std_series * 8) + number_of_samplemix):]
## skip wells for dilution series and sample_mix ##
sample_dest = sample_dest[:number_of_samples]
## cuts off the list after a certain number of samples ##
#### Where is the sample_mix located
sample_mix = plate_96_dil_2.well(sample_mix_well)
#### Where the sample_mix will go in the PCR plate ##
sample_mix_dest = []
sample_mix_column = str(number_std_series + 1)
for well in plate_96_qPCR.columns_by_name()[sample_mix_column]:
sample_mix_dest.append(well)
## PCR will start with dilution series (if desired), followed by an ##
## entire column with sample mix. This is used to normalize between ##
## PCRs ##
sample_mix_dest = sample_mix_dest[:number_of_samplemix]
## Cuts of the list of wells after the desired number of sample mixes ##
# # ALIQUOTING MASTERMIX=======================================================
# # ===========================================================================
##### Variables for volume tracking
start_height_mix = vt.cal_start_height('tube_5mL', start_vol_mix)
current_height = start_height_mix
for i, well in enumerate(MasterMixAliquots):
## aliquot mix, for each well do the following: ##
aspiration_vol = dispension_vol_mix + (dispension_vol_mix / 100 * 2)
## Set correct variables for volume_tracking ##
if i == 0:
p300.pick_up_tip()
## If we are at the first well, start by picking up a tip. ##
elif i % 8 == 0:
p300.drop_tip()
p300.pick_up_tip()
## Then, after every 8th well, drop tip and pick up new ##
current_height, pip_height, bottom_reached = vt.volume_tracking(
'tube_5mL', dispension_vol_mix, 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 ##
p300.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
p300.dispense(dispension_vol_mix, well)
## Dispense the amount specified in dispension_vol to the ##
## location specified in well (looping through plate) ##
p300.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 too many ##
## bubbles) ##
p300.drop_tip()
## when done, drop tip ##
# DISTRIBUTING SAMPLES AND SAMPLE MIX==========================================
# =============================================================================
##### Distributing sample
for sample, destination_well in zip(samples, sample_dest):
## Combine each sample with a destination well ##
p20.pick_up_tip()
## p20 picks up tip from location of specified starting_tip ##
## or following ##
p20.aspirate(sample_volume, sample)
## aspirate sample_volume from sample ##
p20.dispense(sample_volume, destination_well)
## dispense sample_volume in destination_well ##
p20.mix(3, 20, destination_well, rate=5)
## pipette max pipette_volume up&down 3 ##
p20.dispense(20, destination_well)
## alternative to blow_out ##
p20.drop_tip()
## Drop tip in trashbin on 12. ##
##### Distributing sample_mix
for destination_well in sample_mix_dest:
p20.pick_up_tip()
## p20 picks up tip from location of specified tip ##
p20.aspirate(sample_volume, sample_mix)
## aspirate sample_volume from sample ##
p20.dispense(sample_volume, destination_well)
## dispense sample_volume in destination_well ##
p20.mix(3, 20, destination_well, rate=5)
## pipette max pipette_volume up&down 3 ##
p20.dispense(20, destination_well)
## alternative to blow_out ##
p20.drop_tip()
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)
# ----------------------------------------------------------------------------
# =============================================================================
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting Phusion mastermix from a 5 mL tube to 6 PCR strips in
2x 96-wells plates; using volume tracking so that the pipette starts
aspirating at the starting height of the liquid and goes down as the
volume decreases.
Adding primers from PCR strips (with 10 uM primer F&R primer mix)
to PCR strips (with mastermix).
"""
# =============================================================================
# ======================LOADING LABWARE AND PIPETTES===========================
# =============================================================================
## For available labware see "labware/list_of_available_labware". ##
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
3, #deck position
'200tips') #custom name
tips_20 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
4, #deck position
'20tips') #custom name
##### !!! OPTION 1: ROBOT
# mastermix_tube = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware def
# 5, #deck position
# 'mastermix_tube') #custom name
# primer_strips_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 6, #deck position
# 'primer_strips_1') #custom name
# primer_strips_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 7, #deck position
# 'primer_strips_2') #custom name
# mastermix_strips_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 8, #deck position
# 'mastermix_strips_1') #custom name
# mastermix__strips_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 9, #deck position
# 'mastermix_strips_2') #custom name
##### !!! OPTION 2: SIMULATOR
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, #variable derived from opening json
2, #deck position
'mastermix_tube') #custom name
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
primer_strips_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
6, #deck position
'primer_strips_1') #custom name
primer_strips_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
7, #deck position
'primer_strips_2') #custom name
mastermix_strips_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
8, #deck position
'mastermix_strips_1') #custom name
mastermix_strips_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
9, #deck position
'mastermix_strips_2') #custom name
##### Loading pipettes
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
p20 = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=[tips_20]) #assigned tiprack
# =============================================================================
# ==========================VARIABLES TO SET#!!!===============================
# =============================================================================
start_vol = 2060
## The start_vol is the volume (ul) that is in the source labware at ##
## the start of the protocol. ##
dispension_vol = 39
## The dispension_vol is the volume (ul) that needs to be aliquoted ##
## into the destination wells/tubes. ##
primer_vol = 6
## The primer_vol is the volume (ul) of barcoded F&R primer that ##
## needs to be added to the reactions that get a barcode. ##
p300.starting_tip = tips_200.well('A1')
p20.starting_tip = tips_20.well('A1')
## The starting_tip is the location of first pipette tip in the box ##
container = 'tube_5mL'
## The container variable is needed for the volume tracking module. ##
## It tells the module which dimensions to use for the calculations ##
## of the pipette height. It is the source labware from which liquid ##
## is aliquoted. ##
## There are several options to choose from: ##
## 'tube_1.5ml', 'tube_2mL', 'tube_5mL', 'tube_15mL', 'tube_50mL' ##
mastermix_source = mastermix_tube['C1']
# Mastermix destination wells==================================================
mastermix = []
## Create an empty list to append wells to for the mastermix wells. ##
mastermix_columns = ([
mastermix_strips_1.columns_by_name()[column_name]
for column_name in ['2', '7', '11']
] + [
mastermix_strips_2.columns_by_name()[column_name]
for column_name in ['2', '7', '11']
])
## Make a list of columns for the mastermix, this is a list of lists! ##
for column in mastermix_columns:
for well in column:
mastermix.append(well)
## Separate the columns into wells and append them to the empty ##
## mastermix wells list ##
# Primer source tubes==========================================================
primers = []
## Create an empty list to append wells to for the primer wells. ##
primer_columns = ([
primer_strips_1.columns_by_name()[column_name]
for column_name in ['2', '7', '11']
] + [
primer_strips_2.columns_by_name()[column_name]
for column_name in ['2', '7', '11']
])
## Make a list of columns for the primers, this is a list of lists! ##
for column in primer_columns:
for well in column:
primers.append(well)
## Separate the columns into wells and append them to the empty primer##
## wells list ##
# =============================================================================
# =============================================================================
# ==========================PREDIFINED VARIABLES===============================
# =============================================================================
aspiration_vol = dispension_vol + (dispension_vol / 100 * 2)
## The aspiration_vol is the volume (ul) that is aspirated from the ##
## container. ##
##### Variables for volume tracking
start_height = vt.cal_start_height(container, 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. ##
# =============================================================================
# ===============================ALIQUOTING MIX================================
# =============================================================================
## For each column in destination_wells, pick up a tip, than for each ##
## well in these columns pipette mix, and after the+ column drop the tip##
## Repeat untill all columns in the list are done. ##
for i, well in enumerate(mastermix):
## Name all the wells in the plate 'well', for all these do: ##
## If we are at the first well, start by picking up a tip. ##
if i == 0:
p300.pick_up_tip()
## Then, after every 8th well, drop tip and pick up a new one. ##
elif i % 8 == 0:
p300.drop_tip()
p300.pick_up_tip()
current_height, pip_height, bottom_reached = vt.volume_tracking(
container, dispension_vol, current_height)
## The volume_tracking function needs the arguments container ##
## dispension_vol and the current_height which we have set in ##
## this protocol. With those variables, the function updates ##
## the current_height and calculates the delta_height of the ##
## liquid after the next aspiration step. ##
if bottom_reached:
aspiration_location = mastermix_tube['C1'].bottom(z=1) #!!!
protocol.comment("You've reached the bottom!")
else:
aspiration_location = mastermix_source.bottom(pip_height) #!!!
## If the level of the liquid in the next run of the loop will##
## be smaller than 1 we have reached the bottom of the tube. ##
## To prevent the pipette from crashing into the bottom, we ##
## tell it to go home and pause the protocol so that this can ##
## never happen. Set the location of where to aspirate from. ##
## Because we put this in the loop, the location will change ##
## to the newly calculated height after each pipetting step. ##
p300.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
p300.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) ##
p300.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) ##
p300.drop_tip()
# =============================================================================
# ===============================ADDING PRIMERS================================
# =============================================================================
## For the columns in both the source (primers) and the destination ##
## (mix): loop trough the wells in those columns. #
for primer_tube, mix_tube in zip(primers, mastermix):
p20.pick_up_tip()
p20.aspirate(primer_vol, primer_tube)
p20.dispense(primer_vol, mix_tube)
primer_mix_vol = primer_vol + 3
## primer_mix_vol = volume for pipetting up and down ##
p20.mix(3, primer_mix_vol, mix_tube)
p20.dispense(10, mix_tube)
p20.drop_tip()
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix from a 1.5 mL tube to a 96 wells plate;
Adding barcodes from PCR strips to the 96 wells plate.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
## For available labware see "labware/list_of_available_labware". ##
# pipette tips
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
8, #deck position
'20tips_1') #custom name
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
11, #deck position
'20tips_2') #custom name
tips_20_3 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
9, #deck position
'20tips_3') #custom name
if MM_dispension_vol >= 17:
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
10, #deck position
'200tips') #custom name
# Tube_racks & plates
if PCR_tube == 'plate_96':
PCR_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
4, #deck position
'PCR_plate_96') #custom name
elif PCR_tube == 'PCR_strips':
#### !!! OPTION 1: ROBOT
# PCR_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 4, #deck position
# 'PCR_strips_1') #custom name
# if PCR_racks >= 2:
# PCR_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 5, #deck position
# 'PCR_strips_2') #custom name
# if PCR_racks >= 3:
# PCR_3 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 6, #deck position
# 'PCR_strips_3') #custom name
#### !!! OPTION 2: SIMULATOR
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
PCR_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
4, #deck position
'PCR_strips_1') #custom name
if PCR_racks >= 2:
PCR_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
5, #deck position
'PCR_strips_2') #custom name
if PCR_racks >= 3:
PCR_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
6, #deck position
'PCR_strips_3') #custom name
if MastermixWater_tube == 'tube_1.5mL':
MastermixWater = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', #labw def
7, #deck pos
'MastermixWater') #cust name
elif MastermixWater_tube == 'tube_5mL':
##### !!! OPTION 1: ROBOT
# MastermixWater = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware def
# 7, #deck position
# 'MastermixWater') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
MastermixWater = protocol.load_labware_from_definition(
labware_def_5mL, #variable derived from opening json
7, #deck position
'MastermixWater') #custom name
if barcode_tube == 'plate_96':
barcode_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
4, #deck position
'barcode_plate_96') #custom name
elif barcode_tube == 'PCR_strips':
##### !!! OPTION 1: ROBOT
# barcode_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 1, #deck position
# 'barcode_strips_1') #custom name
# if barcode_racks >=2:
# barcode_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 2, #deck position
# 'barcode_strips_2') #custom name
# if barcode_racks >=3:
# barcode_3 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 3, #deck position
# 'barcode_strips_3') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
barcode_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
1, #deck position
'barcode_strips_1') #custom name
if barcode_racks >= 2:
barcode_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
2, #deck position
'barcode_strips_2') #custom name
if barcode_racks >= 3:
barcode_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
3, #deck position
'barcode_strips_3') #custom name
elif barcode_tube == 'tube_1.5mL':
barcode_1 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', #labw def
1, #deck pos
'barcode_rack_1') #cust name
if barcode_racks >= 2:
barcode_2 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', #l def
2, #d pos
'barcode_rack_2') #name
if barcode_racks >= 3:
barcode_3 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', #l def
3, #d pos
'barcode_rack_3') #name
# Pipettes
p20 = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=[tips_20_1, tips_20_2, tips_20_3]) #assigned tiprack
if MM_dispension_vol >= 17:
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
# =============================================================================
# PREDIFINED VARIABLES=========================================================
# =============================================================================
#### MasterMix
MM_aspiration_vol = MM_dispension_vol + (MM_dispension_vol / 100 * 2)
## The aspiration_vol is the volume (ul) that is aspirated from the ##
## container. ##
##### Variables for volume tracking
MM_start_height = vt.cal_start_height(MastermixWater_tube, MM_start_vol)
## Call start height calculation function from volume tracking module. ##
MM_current_height = MM_start_height
## Set the current height to start height at the beginning of the ##
## protocol. ##
barcode_mix_vol = barcode_vol + 3
## barcode_mix_vol = volume for pipetting up and down ##
#### Water
##### Variables for volume tracking
w_start_height = vt.cal_start_height(MastermixWater_tube, w_start_vol)
## Call start height calculation function from volume tracking module. ##
w_current_height = w_start_height
## Set the current height to start height at the beginning of the ##
## protocol. ##
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
##### Setting starting tip
if MM_dispension_vol >= 17: ##
p300.starting_tip = tips_200.well(starting_tip_p200)
p20.starting_tip = tips_20_1.well(starting_tip_p20)
## The starting_tip is the location of first pipette tip in the box ##
##### Tube locations ##
MasterMix = MastermixWater[mastermix_source]
## Location of the tube with mastermix ##
water = MastermixWater[water_source]
## Location of the tube with water ##
#### Where should mastermix go ##
destination_wells = []
if PCR_tube == 'plate_96':
destination_wells = PCR_1.wells()
elif PCR_tube == 'PCR_strips':
PCR_columns = (([
PCR_1.columns_by_name()[column_name]
for column_name in strip_positions
]))
if PCR_racks >= 2:
PCR_columns_2 = (([
PCR_2.columns_by_name()[column_name]
for column_name in strip_positions
]))
PCR_columns = PCR_columns + PCR_columns_2
if PCR_racks >= 3:
PCR_columns_3 = (([
PCR_3.columns_by_name()[column_name]
for column_name in strip_positions
]))
PCR_columns = PCR_columns + PCR_columns_3
for column in PCR_columns:
for well in column:
destination_wells.append(well)
## Make a list with all 96 wells of the plate ##
destination_wells = destination_wells[:number_of_samples]
## cuts off the list after a certain number of wells ##
#### Where are the barcodes located
barcode_wells = []
## Create an empty list to append wells to ##
barcode_columns = (([
barcode_1.columns_by_name()[column_name] for column_name in barcode_loc
]))
if barcode_racks >= 2:
barcode_columns2 = (([
barcode_2.columns_by_name()[column_name]
for column_name in barcode_loc
]))
for column in barcode_columns2:
barcode_columns.append(column)
if barcode_racks >= 3:
barcode_columns3 = (([
barcode_3.columns_by_name()[column_name]
for column_name in barcode_loc
]))
for column in barcode_columns3:
barcode_columns.append(column)
## Make a list of columns, this is a list of lists! ##
for column in barcode_columns:
for well in column:
barcode_wells.append(well)
## Separate the columns into wells and append them to list ##
barcode_wells = barcode_wells[:number_of_samples]
## cuts off the list after a certain number of wells ##
# =============================================================================
## ALIQUOTING MASTERMIX========================================================
## ============================================================================
if MM_dispension_vol < 17:
pipette = p20
else:
pipette = p300
for i, well in enumerate(destination_wells):
## 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 % 16 == 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(
MastermixWater_tube, MM_dispension_vol, MM_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(MM_aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
pipette.dispense(MM_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 BARCODES FOR SAMPLES TO THE MIX=======================================
## ============================================================================
for barcode_well, destination_well in zip(barcode_wells,
destination_wells):
## Loop trough barcode_wells and destination_wells ##
p20.pick_up_tip()
p20.aspirate(barcode_vol, barcode_well)
p20.dispense(barcode_vol, destination_well)
p20.mix(3, barcode_mix_vol, destination_well)
p20.dispense(10, destination_well)
p20.drop_tip()
# =============================================================================
## ADDING VARIABLE VOLUME OF WATER=============================================
## ============================================================================
for water_volume, destination_well in zip(water_µL_list,
destination_wells):
## Loop through water_volumes and destination_well
current_height, pip_height, bottom_reached = vt.volume_tracking(
MastermixWater_tube, water_volume, w_current_height)
## call volume_tracking function, obtain current_height, ##
## pip_height and whether bottom_reached. ##
if bottom_reached:
aspiration_location = water.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 = water.bottom(pip_height)
## Set the location of where to aspirate from. ##
p20.pick_up_tip()
p20.aspirate(water_volume, aspiration_location)
## Aspirate the amount specified in water_volume from the location ##
## specified in aspiration_location. (so a new volume every time ##
## the loop restarts) ##
p20.dispense(water_volume, destination_well)
## Dispense the amount specified in water_volume to the ##
## location specified in well (so a new well and volume every time ##
## the loop restarts) ##
water_mix_volume = water_volume + 3
p20.mix(3, water_mix_volume, destination_well)
## After dispension, mix 3 times with water_volume +3 ##
p20.dispense(10, destination_well)
p20.drop_tip()
def run(protocol: protocol_api.ProtocolContext):
"""
Alliquoting MasterMix
Adding samples from 1.5 mL tubes to PCR_strips or plate.
different DNA volumes + water to add up
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
## For available labware see "labware/list_of_available_labware". ##
#pipette tips
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
9, #deck position
'200tips') #custom name
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
3, #deck position
'20tips_1') #custom name
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
6, #deck position
'20tips_2') #custom name
# Tube_racks & plates
sample_tubes_1 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labware def
1, #deck position
'sample_tubes_1') #custom name
if sample_racks >= 2:
sample_tubes_2 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labw def
4, #deck pos
'sample_tubes_2') #cust name
if sample_racks >= 3:
sample_tubes_3 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labw def
7, #deck pos
'sample_tubes_3') #cust name
if sample_racks >= 4:
sample_tubes_4 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',#labw def
10, #deck pos
'sample_tubes_4') #cust name
if PCR_tubes == 'plate_96':
PCR_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
2, #deck position
'plate_96') #custom name
if PCR_tubes == 'PCR_strips':
### !!! OPTION 1: ROBOT
# PCR_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 2, #deck position
# 'PCR_tube_1') #custom name
# if PCR_racks >= 2:
# PCR_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 5, #deck position
# 'PCR_tube_2') #custom name
# if PCR_racks >= 3:
# PCR_3 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 8, #deck position
# 'PCR_tube_3') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
PCR_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
2, #deck position
'PCR_tube_1') #custom name
if PCR_racks >= 2:
PCR_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
5, #deck position
'PCR_tube_2') #custom name
if PCR_racks >= 3:
PCR_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
8, #deck position
'PCR_tube_2') #custom name
##### !!! OPTION 1: ROBOT
# tubes_5mL = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware def
# 11, #deck position
# 'tubes_5mL') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
tubes_5mL = protocol.load_labware_from_definition(
labware_def_5mL, #variable derived from opening json
11, #deck position
'tubes_5mL') #custom name
# Pipettes
p20 = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=[tips_20_1, tips_20_2]) #assigned tiprack
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
# =============================================================================
# PREDIFINED VARIABLES=========================================================
# =============================================================================
##### Mastermix
aspiration_vol = dispension_vol + (dispension_vol/100*2)
## The aspiration_vol is the volume (ul) that is aspirated from the ##
## container. ##
MM_start_height = vt.cal_start_height('tube_5mL', start_vol_MM)
## Call start height calculation function from volume tracking module. ##
MM_current_height = MM_start_height
## Set the current height to start height at the beginning of the ##
## protocol. ##
##### Water
w_start_height = vt.cal_start_height('tube_5mL', start_vol_w)
## Call start height calculation function from volume tracking module. ##
w_current_height = w_start_height
## Set the current height to start height at the beginning of the ##
## protocol. ##
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
##### Setting starting tip ##
p300.starting_tip = tips_200.well(starting_tip_p200)
p20.starting_tip = tips_20_1.well(starting_tip_p20)
## The starting_tip is the location of first pipette tip in the box ##
# Sample source wells
sample_sources = []
## Create an empty list to append wells to. ##
sample_sources = sample_tubes_1.wells()
if sample_racks >= 2:
sample_sources = sample_sources + sample_tubes_2.wells()
if sample_racks >= 3:
sample_sources = sample_sources + sample_tubes_3.wells()
if sample_racks >= 4:
sample_sources = sample_sources + sample_tubes_4.wells()
sample_sources = sample_sources[:number_of_samples]
# Destination wells
sample_destinations = []
## Create an empty list to append wells to. ##
if PCR_tubes == 'plate_96':
sample_destinations = PCR_1.wells()
elif PCR_tubes == 'PCR_strips':
PCR_columns = (
([PCR_1.columns_by_name()[column_name]
for column_name in strip_positions]))
if PCR_racks >= 2:
PCR_columns_2 = (
([PCR_2.columns_by_name()[column_name]
for column_name in strip_positions]))
PCR_columns = PCR_columns + PCR_columns_2
if PCR_racks >= 3:
PCR_columns_3 = (
([PCR_3.columns_by_name()[column_name]
for column_name in strip_positions]))
PCR_columns = PCR_columns + PCR_columns_3
for column in PCR_columns:
for well in column:
sample_destinations.append(well)
sample_destinations = sample_destinations[:number_of_samples]
##### Tube locations ##
MasterMix = tubes_5mL[mastermix_source]
## Location of the 5mL tube with mastermix ##
water = tubes_5mL[water_tube]
## Location of the 5mL tube with water ##
## ALIQUOTING MASTERMIX========================================================
## ============================================================================
for i, well in enumerate(sample_destinations):
## aliquot mix, for each well do the following: ##
if i == 0:
p300.pick_up_tip()
## If we are at the first well, start by picking up a tip. ##
elif i % 16 == 0:
p300.drop_tip()
p300.pick_up_tip()
## Then, after every 16th well, drop tip and pick up new ##
MM_current_height, pip_height, bottom_reached = vt.volume_tracking(
'tube_5mL', dispension_vol, MM_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 ##
p300.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
p300.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) ##
p300.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) ##
p300.drop_tip()
# =============================================================================
# ADDING SAMPLES AND WATER=====================================================
# =============================================================================
## Loop through source and destination wells
for sample_tube, well, sample_vol in zip(
sample_sources, sample_destinations, DNA_µL_list
):
if sample_vol > 0:
p20.pick_up_tip()
p20.aspirate(sample_vol, sample_tube)
p20.dispense(sample_vol, well)
sample_mix_vol = sample_vol + 3
## mix_vol = volume for pipetting up and down ##
p20.mix(3, sample_mix_vol, well)
p20.dispense(10, well)
p20.drop_tip()
water_vol = max_DNA_volume - sample_vol
## volume of water needed to add a total of max_DNA_volume
if water_vol > 0:
w_current_height, pip_height, bottom_reached = vt.volume_tracking(
'tube_5mL', water_vol, w_current_height)
## call volume_tracking function, obtain current_height, ##
## pip_height and whether bottom_reached. ##
if bottom_reached:
aspiration_location = water.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 = water.bottom(pip_height)
## Set the location of where to aspirate from. ##
p20.pick_up_tip()
p20.aspirate(water_vol, aspiration_location)
p20.dispense(water_vol, well)
mix_vol = water_vol + 3
## mix_vol = volume for pipetting up and down ##
p20.mix(3, mix_vol, well)
p20.dispense(10, well)
p20.drop_tip()
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix from a 1.5 or 5 mL tube to a 96 wells plate or
tube strips that already contain sample.
Mix by pipetting up and down.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
# pipette tips
if MM_dispension_vol > 17:
tips_1 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
7, #deck position
'200tips_1') #custom name
tips_2 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
10, #deck position
'200tips_2') #custom name
else:
tips_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
7, #deck position
'20tips_1') #custom name
tips_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
10, #deck position
'20tips_2') #custom name
# tube racks
if sample_tubes == 'plate_96':
samples_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
1, #deck position
'samples_plate') #custom name
if sample_tubes == 'PCR_strips':
##### !!! OPTION 1: ROBOT
# samples_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 1, #deck position
# 'sample_strips_1') #custom name
# if sample_racks >= 2:
# samples_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 2, #deck position
# 'sample_strips_2') #custom name
# if sample_racks >= 3:
# samples_3 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 3, #deck position
# 'sample_strips_3') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
samples_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from json
1, #deck position
'sample_strips_1') #custom name
if sample_racks >= 2:
samples_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from json
2, #deck position
'sample_strips_2') #custom name
if sample_racks >= 3:
samples_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from json
3, #deck position
'sample_strips_3') #custom name
if MM_tube == 'tube_1.5mL':
MM = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', #labw def
4, #deck pos
'MM_1.5mL') #custname
if MM_tube == 'tube_5mL':
##### !!! OPTION 1: ROBOT
# MM = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware def
# 4, #deck position
# 'mastermix_tube') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
MM = protocol.load_labware_from_definition(
labware_def_5mL, #variable derived from json
4, #deck position
'mastermix_tube') #custom name
# Pipettes
if MM_dispension_vol > 17:
pipette = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_1, tips_2]) #assigned tiprack
else:
pipette = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=[tips_1, tips_2]) #assigned tiprack
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
##### Setting starting tip ##
pipette.starting_tip = tips_1.well(starting_tip)
## The starting_tip is the location of first pipettetip in the first box##
##### Tube locations ##
MasterMix = MM[MM_well]
## Location of the tube with mastermix ##
destination_wells = []
## Where should mastermix go ##
if sample_tubes == 'plate_96':
destination_wells = samples_1.wells()
elif sample_tubes == 'PCR_strips':
sample_columns = (([
samples_1.columns_by_name()[column_name]
for column_name in strip_positions
]))
if sample_racks >= 2:
sample_columns_2 = (([
samples_2.columns_by_name()[column_name]
for column_name in strip_positions
]))
sample_columns = sample_columns + sample_columns_2
if sample_racks >= 3:
sample_columns_3 = (([
samples_3.columns_by_name()[column_name]
for column_name in strip_positions
]))
sample_columns = sample_columns + sample_columns_3
for column in sample_columns:
for well in column:
destination_wells.append(well)
destination_wells = destination_wells[:number_of_samples]
## cuts off the list after a certain number of wells ##
# =============================================================================
# VARIABLES FOR VOLUME TRACKING================================================
# =============================================================================
start_height = vt.cal_start_height(MM_tube, MM_start_vol)
## Call start height calculation function from volume tracking module.##
if MM_start_vol < 500:
start_height = start_height - 8
## start_height is not so good for small amounts
current_height = start_height
## Set the current height to start height at the beginning of the ##
## protocol. ##
# =============================================================================
## ALIQUOTING MASTERMIX========================================================
## ============================================================================
for i, well in enumerate(destination_wells):
## For each destination_well do the following: ##
##### volume tracking ##
current_height, pip_height, bottom_reached = vt.volume_tracking(
MM_tube, MM_dispension_vol, current_height)
## call volume_tracking function, obtain current_height, ##
## pip_height and whether bottom_reached. ##
# What to do if bottom_reached ##
if bottom_reached:
aspiration_location = MasterMix.bottom()
protocol.comment("You've reached the bottom of the tube!")
## If bottom is reached keep pipetting from bottom ##
else:
aspiration_location = MasterMix.bottom(pip_height)
## Set the location of where to aspirate from. ##
##### The actual aliquoting of mastermix ##
pipette.pick_up_tip()
## Pick up a new tip for every sample
pipette.aspirate(MM_dispension_vol, aspiration_location)
## Aspirate the specified volume, from the specified location ##
pipette.dispense(MM_dispension_vol, well)
## Dispense the specified volume to the location specified in ##
## well (so a new well every time the loop restarts) ##
pipette.mix(3, mix_vol, well)
## Mix by pipetting up and down 3x with the specified volume + 3 ##
pipette.dispense(10, well)
## Alternative for blow_out ##
pipette.drop_tip()
## Drop tip in trash ##
# =============================================================================
def run(protocol: protocol_api.ProtocolContext):
"""
Dilute samples in a fixed or varying rates
or transfer samples to different tubes.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
##### Loading pipettes and tips
tips_20 = []
tips_200 = []
if tips_20_needed > 0:
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
11,
'tips_20_1')
tips_20.append(tips_20_1)
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
10,
'tips_20_2')
tips_20.append(tips_20_2)
if tips_20_needed > 96:
tips_20_3 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
7,
'tips_20_3')
tips_20.append(tips_20_3)
if tips_20_needed > 192:
tips_20_4 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul',
8,
'tips_20_4')
tips_20.append(tips_20_4)
elif tips_200_needed > 0:
tips_200_1 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
8,
'tips_200_1')
tips_200.append(tips_200_1)
elif tips_200_needed > 0:
tips_200_1 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
7,
'tips_200_1')
tips_200.append(tips_200_1)
tips_200_2 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
8,
'tips_200_2')
tips_200.append(tips_200_2)
else:
tips_200_1 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
11,
'tips_200_1')
tips_200.append(tips_200_1)
tips_200_2 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
10,
'tips_200_2')
tips_200.append(tips_200_2)
if tips_200_needed > 96:
tips_200_3 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
7,
'tips_200_3')
tips_200.append(tips_200_3)
if tips_200_needed > 192:
tips_200_4 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul',
8,
'tips_200_4')
tips_200.append(tips_200_4)
##### Loading pipettes
if tips_20_needed > 0:
p20 = protocol.load_instrument(
'p20_single_gen2',
'left',
tip_racks = tips_20)
if tips_200_needed > 0:
p300 = protocol.load_instrument(
'p300_single_gen2',
'right',
tip_racks = tips_200)
##### Loading labware
if sample_tubes == 'plate_96':
if sample_racks >= 1:
sample_source_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
1,
'sample_source_1')
if sample_racks >= 2:
sample_source_2 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
4,
'sample_source_2')
if sample_racks >= 3:
sample_source_3 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
2,
'sample_source_3')
if sample_tubes == 'tubes_1.5mL':
if sample_racks >= 1:
sample_source_1 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
1,
'sample_source_1')
if sample_racks >= 2:
sample_source_2 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
2,
'sample_source_2')
if sample_racks >= 3:
sample_source_3 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
4,
'sample_source_3')
if sample_racks >= 4:
sample_source_4 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
5,
'sample_source_4')
if dilution_tubes == 'plate_96':
if dilution_racks >= 1:
dilution_dest_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
6,
'dilution_dest_1')
if dilution_racks >= 2:
dilution_dest_2 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
3,
'dilution_dest_2')
if dilution_racks >= 3:
dilution_dest_3 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr',
5,
'dilution_dest_3')
if dilution_tubes == 'tubes_1.5mL':
if dilution_racks >= 1:
dilution_dest_1 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
6,
'dilution_dest_1')
if dilution_racks >= 2:
dilution_dest_2 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
3,
'dilution_dest_2')
if dilution_racks >= 3:
dilution_dest_3 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
5,
'dilution_dest_3')
if dilution_racks >= 4:
dilution_dest_4 = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
2,
'dilution_dest_4')
if simulate:
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
if sample_tubes == 'PCR_strips':
if sample_racks >= 1:
sample_source_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
1,
'sample_source_1')
if sample_racks >= 2:
sample_source_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
4,
'sample_source_2')
if sample_racks >= 3:
sample_source_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
2,
'sample_source_3')
if sample_racks >= 4:
sample_source_4 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
5,
'sample_source_4')
if dilution_tubes == 'PCR_strips':
if dilution_racks >= 1:
dilution_dest_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
3,
'dilution_dest_1')
if dilution_racks >= 2:
dilution_dest_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
6,
'dilution_dest_2')
if dilution_racks >= 3:
dilution_dest_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
5,
'dilution_dest_3')
if dilution_racks >= 4:
dilution_dest_4 = protocol.load_labware_from_definition(
labware_def_pcrstrips,
2,
'dilution_dest_4')
with open("labware/eppendorfscrewcap_15_tuberack_5000ul/"
"eppendorfscrewcap_15_tuberack_5000ul.json") as labware_file:
labware_def_5mL = json.load(labware_file)
tubes_5mL = protocol.load_labware_from_definition(
labware_def_5mL,
9,
'5mL_tubes')
else:
if sample_tubes == 'PCR_strips':
if sample_racks >= 1:
sample_source_1 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul',
1,
'sample_source_1')
if sample_racks >= 2:
sample_source_2 = protocol.load_labwware(
'pcrstrips_96_wellplate_200ul',
4,
'sample_source_2')
if sample_racks >= 3:
sample_source_3 = protocol.load_labwware(
'pcrstrips_96_wellplate_200ul',
2,
'sample_source_3')
if sample_racks >= 4:
sample_source_4 = protocol.load_labwware(
'pcrstrips_96_wellplate_200ul',
5,
'sample_source_4')
if dilution_tubes == 'PCR_strips':
if dilution_racks >= 1:
dilution_dest_1 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul',
3,
'dilution_dest_1')
if dilution_racks >= 2:
dilution_dest_2 = protocol.load_labwware(
'pcrstrips_96_wellplate_200ul',
6,
'dilution_dest_2')
if dilution_racks >= 3:
dilution_dest_3 = protocol.load_labwware(
'pcrstrips_96_wellplate_200ul',
5,
'dilution_dest_3')
if dilution_racks >= 4:
dilution_dest_4 = protocol.load_labwware(
'pcrstrips_96_wellplate_200ul',
2,
'dilution_dest_4')
tubes_5mL = protocol.load_labware(
'eppendorfscrewcap_15_tuberack_5000ul',
9,
'tubes_5mL')
##### MB: I think this can be removed... but not completely sure because I
#don't know why it was her ein the first place.
# # Setting sample_source
# if sample_racks == 1:
# sample_source_1 = sample_source_1
# elif sample_racks == 2:
# sample_source_1 = sample_source_1
# sample_source_2 = sample_source_2
# elif sample_racks == 3:
# sample_source_1 = sample_source_1
# sample_source_2 = sample_source_2
# sample_source_3 = sample_source_3
# elif sample_racks == 4:
# sample_source_1 = sample_source_1
# sample_source_2 = sample_source_2
# sample_source_3 = sample_source_3
# sample_source_4 = sample_source_4
# # Setting dilution_dest
# if dilution_racks == 1:
# dilution_dest_1 = dilution_dest_1
# elif dilution_racks == 2:
# dilution_dest_1 = dilution_dest_1
# dilution_dest_2 = dilution_dest_2
# elif dilution_racks == 3:
# dilution_dest_1 = dilution_dest_1
# dilution_dest_2 = dilution_dest_2
# dilution_dest_3 = dilution_dest_3
# elif dilution_racks == 4:
# dilution_dest_1 = dilution_dest_1
# dilution_dest_2 = dilution_dest_2
# dilution_dest_3 = dilution_dest_3
# dilution_dest_4 = dilution_dest_4
# =============================================================================
# SETTING LOCATIONS#!!!========================================================
# =============================================================================
##### Setting starting tip
if tips_20_needed > 0:
p20.starting_tip = tips_20_1.well(starting_tip_p20)
if tips_200_needed > 0:
p300.starting_tip = tips_200_1.well(starting_tip_p200)
## The starting_tip is the location of first pipette tip in the box ##
##### Setting tube locations
sample_wells = []
if sample_tubes == 'PCR_strips':
columns_odd = ['1','3','5','7','9','11']
sample_columns = []
if sample_racks >= 1:
sample_columns_1 = (
([sample_source_1.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in sample_columns_1:
sample_columns.append(column)
if sample_racks >= 2:
sample_columns_2 = (
([sample_source_2.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in sample_columns_2:
sample_columns.append(column)
if sample_racks >= 3:
sample_columns_3 = (
([sample_source_3.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in sample_columns_3:
sample_columns.append(column)
if sample_racks >= 4:
sample_columns_4 = (
([sample_source_4.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in sample_columns_4:
sample_columns.append(column)
for column in sample_columns:
for well in column:
sample_wells.append(well)
##makes a list of all wells in 1,2,3 or 4 full plates of PCR strips##
else:
if sample_racks == 1:
for well in sample_source_1.wells():
sample_wells.append(well)
if sample_racks == 2:
for well in sample_source_1.wells():
sample_wells.append(well)
for well in sample_source_2.wells():
sample_wells.append(well)
if sample_racks == 3:
for well in sample_source_1.wells():
sample_wells.append(well)
for well in sample_source_2.wells():
sample_wells.append(well)
for well in sample_source_3.wells():
sample_wells.append(well)
if sample_racks == 4:
for well in sample_source_1.wells():
sample_wells.append(well)
for well in sample_source_2.wells():
sample_wells.append(well)
for well in sample_source_3.wells():
sample_wells.append(well)
for well in sample_source_4.wells():
sample_wells.append(well)
dilution_wells = []
if dilution_tubes == 'PCR_strips':
columns_odd = ['1','3','5','7','9','11']
dilution_columns = []
if dilution_racks >= 1:
dilution_columns_1 = (
([dilution_dest_1.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in dilution_columns_1:
dilution_columns.append(column)
if dilution_racks >= 2:
dilution_columns_2 = (
([dilution_dest_2.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in dilution_columns_2:
dilution_columns.append(column)
if dilution_racks >= 3:
dilution_columns_3 = (
([dilution_dest_3.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in dilution_columns_3:
dilution_columns.append(column)
if dilution_racks >= 4:
dilution_columns_4 = (
([dilution_dest_4.columns_by_name()[column_name]
for column_name in columns_odd]))
for column in dilution_columns_4:
dilution_columns.append(column)
for column in dilution_columns:
for well in column:
dilution_wells.append(well)
##makes a list of all wells in 1,2,3 or 4 full plates of PCR strips##
else:
if dilution_racks == 1:
for well in dilution_dest_1.wells():
dilution_wells.append(well)
if dilution_racks == 2:
for well in dilution_dest_1.wells():
dilution_wells.append(well)
for well in dilution_dest_2.wells():
dilution_wells.append(well)
if dilution_racks == 3:
for well in dilution_dest_1.wells():
dilution_wells.append(well)
for well in dilution_dest_2.wells():
dilution_wells.append(well)
for well in dilution_dest_3.wells():
dilution_wells.append(well)
if dilution_racks == 4:
for well in dilution_dest_1.wells():
dilution_wells.append(well)
for well in dilution_dest_2.wells():
dilution_wells.append(well)
for well in dilution_dest_3.wells():
dilution_wells.append(well)
for well in dilution_dest_4.wells():
dilution_wells.append(well)
sample_wells = sample_wells[:number_of_samples]
dilution_wells = dilution_wells[:number_of_samples]
## cuts off the list after certain number of samples ##
# =============================================================================
# MESSAGE AT THE START=========================================================
# =============================================================================
if len([x for x in water_volumes if x > 0]) > 0:
protocol.pause("I need "+ str(water_tubes) + " tube(s) with 5mL of water.")
# =============================================================================
# ALIQUOTING WATER=============================================================
# =============================================================================
##### Variables for volume tracking and aliquoting
if len([x for x in water_volumes if x > 0]) > 0:
counter = 0 # to count how many tubes already emptied
source = tubes_5mL.wells()[counter]
destination = dilution_wells
container = 'tube_5mL'
start_height = vt.cal_start_height(container, 4800)
current_height = start_height
for i, (well, water_vol) in enumerate(zip(destination, water_volumes)):
## aliquot water in the correct wells, for each well do the following:
if water_vol > 0:
dispension_vol = water_vol
aspiration_vol = dispension_vol + (dispension_vol/100*2)
if i == 0:
if len([x for x in water_volumes[i:i+15] if x < 20]) > 0:
p20.pick_up_tip()
if len([x for x in water_volumes[i:i+15] if x >= 20]) > 0:
p300.pick_up_tip()
## If we are at the first well, start by picking up a tip
elif i % 16 == 0:
try:
p20.drop_tip()
except:
pass
try:
p300.drop_tip()
except:
pass
## Then, after every 16th well, try to drop tip
if len([x for x in water_volumes[i:i+15] if x < 20]) > 0:
p20.pick_up_tip()
if len([x for x in water_volumes[i:i+15] if x >= 20]) > 0:
p300.pick_up_tip()
## Pick up new tip if needed in next 16
current_height, pip_height, bottom_reached = vt.volume_tracking(
container, dispension_vol, current_height)
## call volume_tracking function, obtain current_height,
## pip_height and whether bottom_reached.
if bottom_reached:
## continue with next tube, reset vt
current_height = start_height
current_height, pip_height, bottom_reached = (
vt.volume_tracking(
container, dispension_vol, current_height))
counter = counter + 1
source = tubes_5mL.wells()[counter]
aspiration_location = source.bottom(current_height)
protocol.comment(
"Continue with tube " + str(counter + 1) + " of water")
else:
aspiration_location = source.bottom(pip_height)
## Set the location of where to aspirate from.
if aspiration_vol > 20:
water_pipette = p300
else:
water_pipette = p20
## What pipette to use
#### The actual aliquoting of water
water_pipette.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the
## location specified in aspiration_location.
water_pipette.dispense(dispension_vol, well)
## Dispense the amount specified in dispension_vol to the
## location specified in well (looping through plate)
water_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 too many
## bubbles)
try:
p20.drop_tip()
except:
pass
try:
p300.drop_tip()
except:
pass
## when entire plate is full, try to drop tip
# =============================================================================
# DILUTING SAMPLES=============================================================
# =============================================================================
for sample_well, dilution_well, sample_vol, water_vol in zip(
sample_wells, dilution_wells, sample_volumes, water_volumes):
## Combine each sample with a dilution_well and a destination well ##
if sample_vol > 17:
sample_pipette = p300
else:
sample_pipette = p20
sample_pipette.pick_up_tip()
## p20 picks up tip from location of specified starting_tip ##
## or following ##
sample_pipette.aspirate(sample_vol, sample_well)
## aspirate sample_volume_dil = volume for dilution from sample ##
sample_pipette.dispense(sample_vol, dilution_well)
## dispense sample_volume_dil = volume for dilution into dil_well ##
if water_vol > 0:
sample_pipette.mix(3, sample_vol + 3, dilution_well)
## pipette up&down 3x to get everything from the tip ##
sample_pipette.dispense(20, dilution_well)
## instead of blow-out
sample_pipette.drop_tip()
## Drop tip in trashbin on 12. ##
# =============================================================================
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix;
Adding barcoded primers.
"""
# =============================================================================
# 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', 2,
'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', 2,
'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
plate_96 = protocol.load_labware('biorad_96_wellplate_200ul_pcr', 6,
'plate_96')
if qPCR:
big_primer_source = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', 8,
'big_primer_source')
if mastermix_tube_type == 'tube_1.5mL':
mastermix_tube = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', 3,
'mastermix_tube')
if primer_tube_type == 'plate_96':
primer_source_1 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', 1, 'primer_source_1')
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, 3, 'mastermix_tube')
if primer_tube_type == 'PCR_strips':
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
primer_source_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, 4, 'primer_source_1')
if primer_racks >= 2:
primer_source_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, 1, 'primer_source_2')
if primer_racks >= 3:
primer_source_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, 11, 'primer_source_3')
else: #Robot
if mastermix_tube_type == 'tube_5mL':
mastermix_tube = protocol.load_labware(
'eppendorfscrewcap_15_tuberack_5000ul', 3, 'mastermix_tube')
if primer_tube_type == 'PCR_strips':
primer_source_1 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul', 4, 'primer_source_1')
if primer_racks >= 2:
primer_source_2 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul', 1, 'primer_source_2')
if primer_racks >= 3:
primer_source_3 = protocol.load_labware(
'pcrstrips_96_wellplate_200ul', 11, 'primer_source_3')
# 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.
primer_mix_vol = primer_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 with all 96 wells of the plate
wells = []
for well in plate_96.wells():
wells.append(well)
# Create the list of wells where samples should go
sample_wells = wells[:number_of_primers]
## cuts off the list after a the number_of_samples number of wells
# Create the list of wells where standard sample replicates should go
slice_std_wells = slice(number_of_primers,
number_of_primers + number_of_std_samples)
## Slice the list - I need the number_of_std_samples of wells after
## the number_of_samples wells. So I need a certain amount of wells
## after the sample wells, but not all of the wells after that.
## This slices the list after the samples and after the std sample
## so that we only take the wells in between.
std_sample_wells = wells[slice_std_wells]
# Create the list of wells where standerd series should go
std_series_wells = []
std_series_columns = ([
plate_96.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)
# Add the wells of the standards into 1 list
## This needs to be a list separate from the rest of the wells because
## they need the same primer from a separate tube.
std_wells = std_sample_wells + std_series_wells
# Add all the wells that need mastermix into 1 list
MasterMixAliquots = sample_wells + std_wells
# Primer locations
primer_wells = []
if primer_tube_type == 'PCR_strips':
primer_columns = []
if primer_racks >= 1:
primer_columns_1 = (([
primer_source_1.columns_by_name()[column_name]
for column_name in primer_loc
]))
for column in primer_columns_1:
primer_columns.append(column)
if primer_racks >= 2:
primer_columns_2 = (([
primer_source_2.columns_by_name()[column_name]
for column_name in primer_loc
]))
for column in primer_columns_2:
primer_columns.append(column)
if primer_racks >= 3:
primer_columns_3 = (([
primer_source_3.columns_by_name()[column_name]
for column_name in primer_loc
]))
for column in primer_columns_3:
primer_columns.append(column)
## Make a list of columns, this is a list of lists!
for column in primer_columns:
for well in column:
primer_wells.append(well)
if primer_tube_type == 'plate_96':
if primer_racks == 1:
for well in primer_source_1.wells():
primer_wells.append(well)
if primer_racks == 2:
for well in primer_source_2.wells():
primer_wells.append(well)
if primer_racks == 3:
for well in primer_source_3.wells():
primer_wells.append(well)
primer_wells = primer_wells[:number_of_primers]
# Set location for primer for standards
if number_of_std_samples >= 1:
std_primer = big_primer_source.wells_by_name()[std_primer_loc]
# =============================================================================
## 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 PRIMERS FOR SAMPLES TO THE MIX---------------------------------------
for primer_well, sample_well in zip(primer_wells, sample_wells):
## Loop trough primer_wells and sample_wells
p20.pick_up_tip()
p20.aspirate(primer_vol, primer_well)
p20.dispense(primer_vol, sample_well)
p20.mix(3, primer_mix_vol, sample_well)
p20.dispense(10, sample_well)
p20.drop_tip()
## ----------------------------------------------------------------------------
## ADDING PRIMERS FOR STANDARDS TO THE MIX-------------------------------------
for well in std_wells:
p20.pick_up_tip()
p20.aspirate(primer_vol, std_primer)
## use last primer pair for NTC and std series
p20.dispense(primer_vol, well)
p20.mix(3, primer_mix_vol, well)
p20.dispense(10, well)
p20.drop_tip()
## ----------------------------------------------------------------------------
## LIGHTS----------------------------------------------------------------------
if not qPCR:
protocol.set_rail_lights(False)
# ----------------------------------------------------------------------------
## ============================================================================
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix from a 5 mL tube to a 96 wells plate or tube strips.
Followed by adding samples.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
## For available labware see "labware/list_of_available_labware". ##
# pipette tips
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
10, #deck position
'200tips') #custom name
# Tube_racks & plates
if PCR_tubes == 'plate_96':
PCR_tube = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
8, #deck position
'plate_96') #custom name
if PCR_tubes == 'PCR_strips':
##### !!! OPTION 1: ROBOT
# PCR_tube_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 8, #deck position
# 'PCR_tube_1') #custom name
# if PCR_racks >= 2:
# PCR_tube_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 5, #deck position
# 'PCR_tube_2') #custom name
# if PCR_racks >= 3:
# PCR_tube_3 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 2, #deck position
# 'PCR_tube_3') #custom name
# if PCR_racks == 4:
# PCR_tube_4 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 1, #deck position
# 'PCR_tube_4') #custom name
##### !!! OPTION 2: SIMULATOR
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
PCR_tube_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
8, #deck position
'PCR_tube_1') #custom name
if PCR_racks >= 2:
PCR_tube_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
5, #deck position
'PCR_tube_2') #custom name
if PCR_racks >= 3:
PCR_tube_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
2, #deck position
'PCR_tube_3') #custom name
if PCR_racks >= 4:
PCR_tube_4 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
1, #deck position
'PCR_tube_4') #custom name
##### !!! OPTION 1: ROBOT
# mastermix_tube = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul', #labware def
# 4, #deck position
# 'mastermix_tube') #custom name
##### !!! OPTION 2: SIMULATOR
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, #variable derived from opening json
4, #deck position
'mastermix_tube') #custom name
# Pipettes
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
# =============================================================================
# PREDIFINED VARIABLES=========================================================
# =============================================================================
aspiration_vol = dispension_vol + (dispension_vol / 100 * 2)
## The aspiration_vol is the volume (ul) that is aspirated from the ##
## container. ##
##### Variables for volume tracking
start_height = vt.cal_start_height('tube_5mL', 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. ##
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
##### Setting starting tip ##
p300.starting_tip = tips_200.well(starting_tip_p200)
## The starting_tip is the location of first pipette tip in the box ##
##### Tube locations ##
MasterMix = mastermix_tube[mastermix_source]
## Location of the 5mL tube with mastermix ##
#### Where should mastermix go ##
number_of_wells = number_of_samples + number_of_NTCs
##How many wells do need to be filled with mastermix ##
MasterMixAliquots = []
if PCR_tubes == 'plate_96':
MasterMixAliquots = PCR_tube.wells()
elif PCR_tubes == 'PCR_strips':
PCR_columns = (([
PCR_tube_1.columns_by_name()[column_name]
for column_name in strip_positions
]))
if PCR_racks >= 2:
PCR_columns_2 = (([
PCR_tube_2.columns_by_name()[column_name]
for column_name in strip_positions
]))
PCR_columns = PCR_columns + PCR_columns_2
if PCR_racks >= 3:
PCR_columns_3 = (([
PCR_tube_3.columns_by_name()[column_name]
for column_name in strip_positions
]))
PCR_columns = PCR_columns + PCR_columns_3
if PCR_racks >= 4:
PCR_columns_4 = (([
PCR_tube_4.columns_by_name()[column_name]
for column_name in strip_positions
]))
PCR_columns = PCR_columns + PCR_columns_4
for column in PCR_columns:
for well in column:
MasterMixAliquots.append(well)
## Make a list with all wells for PCR ##
MasterMixAliquots = MasterMixAliquots[:number_of_wells]
## cuts off the list after a certain number of wells ##
# =============================================================================
## ALIQUOTING MASTERMIX========================================================
## ============================================================================
for i, well in enumerate(MasterMixAliquots):
## aliquot mix, for each well do the following: ##
if i == 0:
p300.pick_up_tip()
## If we are at the first well, start by picking up a tip. ##
elif i % 16 == 0:
p300.drop_tip()
p300.pick_up_tip()
## Then, after every 16th well, drop tip and pick up new ##
current_height, pip_height, bottom_reached = vt.volume_tracking(
'tube_5mL', 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 ##
p300.aspirate(aspiration_vol, aspiration_location)
## Aspirate the amount specified in aspiration_vol from the ##
## location specified in aspiration_location. ##
p300.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) ##
p300.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) ##
p300.drop_tip()
def run(protocol: protocol_api.ProtocolContext):
"""
Aliquoting mastermix from a 5 mL tube to a 96 wells plate;
Adding primers from PCR strips (with 10 uM primer F&R primer mix) to
the 96 wells plate.
"""
# =============================================================================
# LOADING LABWARE AND PIPETTES=================================================
# =============================================================================
## For available labware see "labware/list_of_available_labware". ##
# pipette tips
if dispension_vol > 19:
tips_200 = protocol.load_labware(
'opentrons_96_filtertiprack_200ul', #labware definition
2, #deck position
'200tips') #custom name
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
7, #deck position
'20tips_1') #custom name
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
10, #deck position
'20tips_2') #custom name
tips_20 = [tips_20_1, tips_20_2]
else:
tips_20_1 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
2, #deck position
'20tips_1') #custom name
tips_20_2 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
7, #deck position
'20tips_2') #custom name
tips_20_3 = protocol.load_labware(
'opentrons_96_filtertiprack_20ul', #labware definition
10, #deck position
'20tips_3') #custom name
tips_20 = [tips_20_1, tips_20_2, tips_20_3]
# Tube_racks & plates
plate_96 = protocol.load_labware(
'biorad_96_wellplate_200ul_pcr', #labware definition
6, #deck position
'plate_96') #custom name
if mastermix_tube_type == 'tube_1.5mL':
mastermix_tube = protocol.load_labware(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
3,
'mastermix_tube')
# ##### !!! OPTION 1: ROBOT
# if mastermix_tube_type == 'tube_5mL':
# mastermix_tube = protocol.load_labware(
# 'eppendorfscrewcap_15_tuberack_5000ul',#labware def
# 3, #deck position
# 'mastermix_tube') #custom name
# primer_strips_1 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 4, #deck position
# 'primer_strips_1') #custom name
# if primer_racks >=2:
# primer_strips_2 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 1, #deck position
# 'primer_strips_2') #custom name
# if primer_racks >=3:
# primer_strips_3 = protocol.load_labware(
# 'pcrstrips_96_wellplate_200ul', #labware definition
# 11, #deck position
# 'primer_strips_3') #custom name
##### !!! OPTION 2: 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, #variable derived from opening json
3, #deck position
'mastermix_tube') #custom name
with open("labware/pcrstrips_96_wellplate_200ul/"
"pcrstrips_96_wellplate_200ul.json") as labware_file:
labware_def_pcrstrips = json.load(labware_file)
primer_strips_1 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
4, #deck position
'primer_strips_1') #custom name
if primer_racks >=2:
primer_strips_2 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
1, #deck position
'primer_strips_2') #custom name
if primer_racks >=3:
primer_strips_3 = protocol.load_labware_from_definition(
labware_def_pcrstrips, #variable derived from opening json
11, #deck position
'primer_strips_3') #custom name
# Pipettes
if dispension_vol > 19:
p300 = protocol.load_instrument(
'p300_single_gen2', #instrument definition
'right', #mount position
tip_racks=[tips_200]) #assigned tiprack
p20 = protocol.load_instrument(
'p20_single_gen2', #instrument definition
'left', #mount position
tip_racks=tips_20) #assigned tiprack
# =============================================================================
# PREDIFINED VARIABLES=========================================================
# =============================================================================
aspiration_vol = dispension_vol + (dispension_vol/100*2)
## The aspiration_vol is the volume (ul) 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. ##
primer_mix_vol = primer_vol + 3
## primer_mix_vol = volume for pipetting up and down ##
# =============================================================================
# SETTING LOCATIONS============================================================
# =============================================================================
##### Setting starting tip ##
if dispension_vol > 19:
p300.starting_tip = tips_200.well(starting_tip_p200)
p20.starting_tip = tips_20_1.well(starting_tip_p20)
## The starting_tip is the location of first pipette tip in the box ##
##### Tube locations ##
MasterMix = mastermix_tube[mastermix_source]
## Location of the 5mL tube with mastermix ##
#### Where should mastermix go ##
number_of_wells = number_of_primers
##How many wells do need to be filled with mastermix ##
sample_wells = []
for well in plate_96.wells():
sample_wells.append(well)
## Make a list with all 96 wells of the plate ##
sample_wells = sample_wells[:number_of_wells]
## cuts off the list after a certain number of wells ##
standard_wells = []
if number_std_series > 0:
std_series_columns = (
[plate_96.columns_by_name()[column_name] for column_name in
['12', '11', '10']])
std_series_columns = std_series_columns[:number_std_series]
## reserve a column at the end of the plate for every std_series
for column in std_series_columns:
column = column[:length_std_series]
for well in column:
standard_wells.append(well)
## cut off the columns after a certain std_series length
MasterMixAliquots = sample_wells + standard_wells
#### Where are the primers located
primer_wells = []
## Create an empty list to append wells to ##
primer_columns = (
([primer_strips_1.columns_by_name()[column_name]
for column_name in primer_loc]))
if primer_racks >= 2:
primer_columns2 = (
([primer_strips_2.columns_by_name()[column_name]
for column_name in primer_loc]))
for column in primer_columns2:
primer_columns.append(column)
if primer_racks >= 3:
primer_columns3 = (
([primer_strips_3.columns_by_name()[column_name]
for column_name in primer_loc]))
for column in primer_columns3:
primer_columns.append(column)
## Make a list of columns, this is a list of lists! ##
for column in primer_columns:
for well in column:
primer_wells.append(well)
primer_wells = primer_wells[:number_of_primers]
## Separate the columns into wells and append them to list ##
# =============================================================================
## 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 PRIMERS FOR SAMPLES TO THE MIX=======================================
## ============================================================================
for primer_well, sample_well in zip(primer_wells, sample_wells):
## Loop trough primer_wells and sample_wells ##
p20.pick_up_tip()
p20.aspirate(primer_vol, primer_well)
p20.dispense(primer_vol, sample_well)
p20.mix(3, primer_mix_vol, sample_well)
p20.dispense(10, sample_well)
p20.drop_tip()
# =============================================================================
## ADDING PRIMERS FOR STD SERIES TO THE MIX====================================
## ============================================================================
for well in standard_wells:
p20.pick_up_tip()
p20.aspirate(primer_vol, primer_wells[-1])
## use last primer pair for NTC and std series
p20.dispense(primer_vol, well)
p20.mix(3, primer_mix_vol, well)
p20.dispense(10, well)
p20.drop_tip()