config.trash_control = True
stock_volume = 630
########################################################################################################################
# Labware
########################################################################################################################
# Configure the tips
tips10 = labware_manager.load('opentrons_96_tiprack_10ul', 1, label='tips10')
tips300a = labware_manager.load('opentrons_96_tiprack_300ul',
4,
label='tips300a')
# Configure the pipettes.
p10 = instruments_manager.P10_Single(mount='left', tip_racks=[tips10])
p50 = instruments_manager.P50_Single(mount='right', tip_racks=[tips300a])
# Control tip usage
p10.start_at_tip(tips10[p10_start_tip])
p50.start_at_tip(tips300a[p50_start_tip])
# All the labware containers
eppendorf_1_5_rack = labware_manager.load(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
5,
label='eppendorf_1_5_rack')
plateA = labware_manager.load('biorad_96_wellplate_200ul_pcr',
6,
label='plateA')
plateB = labware_manager.load('biorad_96_wellplate_200ul_pcr',
def run(protocol_context: protocol_api.ProtocolContext):
config.protocol_context = protocol_context
####################################################################################################################
## Well volumes
####################################################################################################################
strand_volume_min = 0
strand_volume_max = 28
strand_a_volume_count = 6
strand_b_volume_count = 5
# historic : strand_volumes = [0, 2, 5, 8, 12, 16, 20, 28]
strand_a_volumes = list(
float_range(strand_volume_min, strand_volume_max,
strand_a_volume_count))
strand_b_volumes = list(
float_range(strand_volume_min, strand_volume_max,
strand_b_volume_count))
replica_groups = list(
pair_up_strand_volumes(strand_a_volumes, strand_b_volumes))
replica_groups.append(
make_pair(mean(strand_a_volumes[-2], strand_a_volumes[-3]),
mean(strand_b_volumes[-2], strand_b_volumes[-3])))
replica_groups.append(
make_pair(mean(strand_a_volumes[-1], strand_a_volumes[-2]),
mean(strand_b_volumes[-1], strand_b_volumes[-2])))
####################################################################################################################
## Accounting
####################################################################################################################
num_columns = 12
num_rows = 8
num_wells = num_rows * num_columns
num_replicates = 3
num_replica_groups = num_wells // num_replicates
num_replica_groups_per_row = num_columns // num_replicates
max_dna_working_concentration = Concentration(0.5, "uM")
nominal_source_strand_concentration = Concentration(10, "uM")
buffer_source_concentration = Concentration(5, "x")
evagreen_source_concentration = Concentration(
25, "uM") # per https://biotium.com/product/evagreen-dye-20x-in-water/
evagreen_target_concentration = max_dna_working_concentration * 2 # we figure we should always have more evagreen than reagents
total_well_volume = 84
buffer_per_well = total_well_volume / buffer_source_concentration.x
evagreen_per_well = total_well_volume / evagreen_source_concentration.molar * evagreen_target_concentration.molar
dna_and_water_per_well = total_well_volume - buffer_per_well - evagreen_per_well
assert len(replica_groups) == num_replica_groups
####################################################################################################################
## Strand Dilution
####################################################################################################################
# Diluting each strand
strand_dilution_vol = 1200
strand_dilution_factor = strand_volume_max / total_well_volume * nominal_source_strand_concentration.molar / max_dna_working_concentration.molar
strand_dilution_source_vol = strand_dilution_vol / strand_dilution_factor
strand_dilution_water_vol = strand_dilution_vol - strand_dilution_source_vol
####################################################################################################################
## Plating Volumes
####################################################################################################################
# Figure out the strand a, strand b, and per-well-water volumes
def make_empty_plate():
row = [0] * num_columns
result = []
for i in range(num_rows):
result.append(row.copy())
return result
strand_a_plate = make_empty_plate()
strand_b_plate = make_empty_plate()
for i, replica_group in enumerate(replica_groups):
row = i // num_replica_groups_per_row
col_first = (i % num_replica_groups_per_row) * num_replicates
for j in range(num_replicates):
col = col_first + j
strand_a_plate[row][col] = replica_group['StrandA']
strand_b_plate[row][col] = replica_group['StrandB']
total_water_plate = make_empty_plate()
common_water_per_well = infinity
for row in range(num_rows):
for col in range(num_columns):
total_water_plate[row][
col] = total_well_volume - buffer_per_well - evagreen_per_well - strand_a_plate[
row][col] - strand_b_plate[row][col]
common_water_per_well = min(common_water_per_well,
total_water_plate[row][col])
master_mix_per_well = buffer_per_well + evagreen_per_well + common_water_per_well
master_mix_plate = make_empty_plate()
for row in range(num_rows):
for col in range(num_columns):
master_mix_plate[row][col] = master_mix_per_well
per_well_water_plate = make_empty_plate()
for row in range(num_rows):
for col in range(num_columns):
per_well_water_plate[row][
col] = dna_and_water_per_well - common_water_per_well - strand_a_plate[
row][col] - strand_b_plate[row][col]
####################################################################################################################
## Master Mix
####################################################################################################################
master_mix_buffer_nominal_vol = num_wells * buffer_per_well
master_mix_evagreen_nominal_vol = num_wells * evagreen_per_well
mm_overhead_factor = 1.1
master_mix_buffer_vol = master_mix_buffer_nominal_vol * mm_overhead_factor
master_mix_evagreen_vol = master_mix_evagreen_nominal_vol * mm_overhead_factor
master_mix_common_water_vol = num_wells * common_water_per_well * mm_overhead_factor
master_mix_vol = master_mix_buffer_vol + master_mix_evagreen_vol + master_mix_common_water_vol
####################################################################################################################
# Labware
####################################################################################################################
# Configure the tips
tips300a = labware_manager.load('opentrons_96_tiprack_300ul',
slot=1,
label='tips300a')
tips10 = labware_manager.load('opentrons_96_tiprack_10ul',
slot=4,
label='tips10')
tips300b = labware_manager.load('opentrons_96_tiprack_300ul',
slot=7,
label='tips300b')
# Configure the pipettes.
p10 = instruments_manager.P10_Single(mount='left', tip_racks=[tips10])
p50 = instruments_manager.P50_Single(mount='right',
tip_racks=[tips300a, tips300b])
# Blow out faster than default in an attempt to avoid hanging droplets on the pipettes after blowout (probably not needed any more)
p10.set_flow_rate(blow_out=p10.get_flow_rates()['blow_out'] *
config.blow_out_rate_factor)
p50.set_flow_rate(blow_out=p50.get_flow_rates()['blow_out'] *
config.blow_out_rate_factor)
# Control tip usage
p10.start_at_tip(tips10[p10_start_tip])
p50.start_at_tip(tips300a[p50_start_tip])
# Labware containers
eppendorf_1_5_rack = labware_manager.load(
'opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap',
slot=5,
label='eppendorf_1_5_rack')
plate = labware_manager.load('biorad_96_wellplate_200ul_pcr',
slot=6,
label='plate')
# Name specific places in the labware containers
diluted_strand_a = eppendorf_1_5_rack['A6']
diluted_strand_b = eppendorf_1_5_rack['B6']
eppendorf_5_0_rack = labware_manager.load(
'Atkinson_15_tuberack_5ml_eppendorf',
slot=8,
label='eppendorf_5_0_rack')
master_mix = eppendorf_5_0_rack['A1']
waterA = eppendorf_5_0_rack['C4']
waterB = eppendorf_5_0_rack['C5']
note_liquid(location=waterA, name='Water', initially=waterA_initial_volume)
note_liquid(location=waterB, name='Water', initially=waterB_initial_volume)
####################################################################################################################
# Well & Pipettes
####################################################################################################################
# Figuring out what pipettes should pipette what volumes
p10_max_vol = 10
p50_min_vol = 5
def usesP10(volume, allow_zero=False):
return (allow_zero or 0 < volume) and (volume < p50_min_vol
and volume <= p10_max_vol)
####################################################################################################################
# Making master mix and diluting strands
####################################################################################################################
def diluteStrands():
if manually_dilute_strands:
note_liquid(location=diluted_strand_a,
name='Diluted StrandA',
initially=strand_dilution_vol)
note_liquid(location=diluted_strand_b,
name='Diluted StrandB',
initially=strand_dilution_vol)
log('Diluting Strands')
info(
pretty.format(
'Diluted Strand A recipe: water={0:n} strandA={1:n} vol={2:n}',
strand_dilution_water_vol, strand_dilution_source_vol,
strand_dilution_vol))
info(
pretty.format(
'Diluted Strand B recipe: water={0:n} strandB={1:n} vol={2:n}',
strand_dilution_water_vol, strand_dilution_source_vol,
strand_dilution_vol))
user_prompt('Ensure diluted strands manually present and mixed')
else:
strand_a = eppendorf_1_5_rack['A1']
strand_b = eppendorf_1_5_rack['B1']
assert strand_a_min_vol >= strand_dilution_source_vol + strand_a.geometry.min_aspiratable_volume
assert strand_b_min_vol >= strand_dilution_source_vol + strand_b.geometry.min_aspiratable_volume
note_liquid(location=strand_a,
name='StrandA',
concentration=strand_a_conc,
initially_at_least=strand_a_min_vol
) # i.e.: we have enough, just not specified how much
note_liquid(location=strand_b,
name='StrandB',
concentration=strand_b_conc,
initially_at_least=strand_b_min_vol) # ditto
note_liquid(location=diluted_strand_a, name='Diluted StrandA')
note_liquid(location=diluted_strand_b, name='Diluted StrandB')
# We used to auto-mix, but now, even when auto-diluting, we rely on user to have mixed on the vortexer
# p50.layered_mix([strand_a])
# p50.layered_mix([strand_b])
# Create dilutions of strands
log('Moving water for diluting Strands A and B')
p50.transfer(
strand_dilution_water_vol,
waterA,
[diluted_strand_a, diluted_strand_b],
new_tip=
'once', # can reuse for all diluent dispensing since dest tubes are initially empty
trash=config.trash_control)
log('Diluting Strand A')
p50.transfer(strand_dilution_source_vol,
strand_a,
diluted_strand_a,
trash=config.trash_control,
keep_last_tip=True)
p50.layered_mix([diluted_strand_a])
log('Diluting Strand B')
p50.transfer(strand_dilution_source_vol,
strand_b,
diluted_strand_b,
trash=config.trash_control,
keep_last_tip=True)
p50.layered_mix([diluted_strand_b])
def createMasterMix():
if manually_make_master_mix:
note_liquid(location=master_mix,
name='Master Mix',
initially=master_mix_vol)
log('Creating Master Mix')
info(
pretty.format(
'Master Mix recipe: water={0:n} buffer={1:n} EvaGreen={2:n} total={3:n} (extra={4}%)',
master_mix_common_water_vol, master_mix_buffer_vol,
master_mix_evagreen_vol, master_mix_vol,
100.0 * (mm_overhead_factor - 1)))
user_prompt('Ensure master mix manually present and mixed')
else:
# Mostly just for fun, we put the ingredients for the master mix in a nice warm place to help them melt
temp_slot = 11
temp_module = modules_manager.load('tempdeck', slot=temp_slot)
screwcap_rack = labware_manager.load(
'opentrons_24_aluminumblock_generic_2ml_screwcap',
slot=temp_slot,
label='screwcap_rack',
share=True,
well_geometry=IdtTubeWellGeometry)
buffers = list(zip(screwcap_rack.rows(0), buffer_volumes))
evagreens = list(zip(screwcap_rack.rows(1), evagreen_volumes))
for buffer in buffers:
note_liquid(location=buffer[0],
name='Buffer',
initially=buffer[1],
concentration=buffer_source_concentration)
for evagreen in evagreens:
note_liquid(location=evagreen[0],
name='Evagreen',
initially=evagreen[1],
concentration=evagreen_source_concentration)
note_liquid(location=master_mix, name='Master Mix')
# Buffer was just unfrozen. Mix to ensure uniformity. EvaGreen doesn't freeze, no need to mix
p50.layered_mix([buffer for buffer, __ in buffers], incr=2)
# transfer from multiple source wells, each with a current defined volume
def transfer_multiple(msg, xfer_vol_remaining, tubes, dest,
new_tip, *args, **kwargs):
tube_index = 0
cur_well = None
cur_vol = 0
min_vol = 0
while xfer_vol_remaining > 0:
if xfer_vol_remaining < p50_min_vol:
warn(
"remaining transfer volume of %f too small; ignored"
% xfer_vol_remaining)
return
# advance to next tube if there's not enough in this tube
while cur_well is None or cur_vol <= min_vol:
if tube_index >= len(tubes):
fatal('%s: more reagent needed' % msg)
cur_well = tubes[tube_index][0]
cur_vol = tubes[tube_index][1]
min_vol = max(
p50_min_vol,
cur_vol / config.
min_aspirate_factor_hack, # tolerance is proportional to specification of volume. can probably make better guess
cur_well.geometry.min_aspiratable_volume)
tube_index = tube_index + 1
this_vol = min(xfer_vol_remaining, cur_vol - min_vol)
assert this_vol >= p50_min_vol # TODO: is this always the case?
log('%s: xfer %f from %s in %s to %s in %s' %
(msg, this_vol, cur_well, cur_well.parent, dest,
dest.parent))
p50.transfer(this_vol,
cur_well,
dest,
trash=config.trash_control,
new_tip=new_tip,
**kwargs)
xfer_vol_remaining -= this_vol
cur_vol -= this_vol
def mix_master_mix():
log('Mixing Master Mix')
p50.layered_mix(
[master_mix],
incr=2,
initial_turnover=master_mix_evagreen_vol * 1.2,
max_tip_cycles=config.layered_mix.max_tip_cycles_large)
log('Creating Master Mix: Water')
p50.transfer(master_mix_common_water_vol,
waterB,
master_mix,
trash=config.trash_control)
log('Creating Master Mix: Buffer')
transfer_multiple(
'Creating Master Mix: Buffer',
master_mix_buffer_vol,
buffers,
master_mix,
new_tip='once',
keep_last_tip=True
) # 'once' because we've only got water & buffer in context
p50.done_tip() # EvaGreen needs a new tip
log('Creating Master Mix: EvaGreen')
transfer_multiple(
'Creating Master Mix: EvaGreen',
master_mix_evagreen_vol,
evagreens,
master_mix,
new_tip='always',
keep_last_tip=True
) # 'always' to avoid contaminating the Evagreen source w/ buffer
mix_master_mix()
########################################################################################################################
# Plating
########################################################################################################################
def plateMasterMix():
log('Plating Master Mix')
for row in range(num_rows):
for col in range(num_columns):
volume = master_mix_plate[row][col]
if volume == 0: continue
p: EnhancedPipetteV1 = p10 if usesP10(volume) else p50
if not p.tip_attached:
p.pick_up_tip()
well = plate.rows(row).wells(col)
p.transfer(volume,
master_mix,
well,
new_tip='never',
trash=config.trash_control,
full_dispense=True)
p10.done_tip()
p50.done_tip()
def platePerWellWater():
log('Plating per-well water')
# Plate per-well water. We save tips by being happy to pollute our water trough with a bit of master mix.
for row in range(num_rows):
for col in range(num_columns):
volume = per_well_water_plate[row][col]
if volume == 0: continue
p: EnhancedPipetteV1 = p10 if usesP10(volume) else p50
if not p.tip_attached:
p.pick_up_tip()
well = plate.rows(row).wells(col)
p.transfer(volume,
waterB,
well,
new_tip='never',
trash=config.trash_control,
full_dispense=True)
p10.done_tip()
p50.done_tip()
def plateStrandA():
# Plate strand A
# All plate wells at this point only have water and master mix, so we can't get cross-plate-well
# contamination. We only need to worry about contaminating the Strand A source, which we accomplish
# by using new_tip='always'. Update: we don't worry about that pollution, that source is disposable.
# So we can minimize tip usage.
log('Plating Strand A')
for row in range(num_rows):
for col in range(num_columns):
volume = strand_a_plate[row][col]
if volume == 0: continue
p: EnhancedPipetteV1 = p10 if usesP10(volume) else p50
if not p.tip_attached:
p.pick_up_tip()
well = plate.rows(row).wells(col)
log('Plating Strand A: volume %d with %s' % (volume, p.name))
p.transfer(volume,
diluted_strand_a,
well,
new_tip='never',
trash=config.trash_control,
full_dispense=True)
p10.done_tip()
p50.done_tip()
def mix_plate_well(well, keep_last_tip=False):
p50.layered_mix([well], incr=0.75, keep_last_tip=keep_last_tip)
def plateStrandBAndMix():
# Plate strand B and mix
# Mixing always needs the p50, but plating may need either; optimize tip usage
log('Plating Strand B')
mixed_wells = set()
for row in range(num_rows):
for col in range(num_columns):
volume = strand_b_plate[row][col]
if volume == 0: continue
p: EnhancedPipetteV1 = p10 if usesP10(volume,
allow_zero=True) else p50
well = plate.rows(row).wells(col)
log("Plating Strand B: well='%s' vol=%d pipette=%s" %
(well.get_name(), volume, p.name))
if not p.tip_attached: p.pick_up_tip()
p.transfer(volume,
diluted_strand_b,
well,
new_tip='never',
trash=config.trash_control,
full_dispense=True)
if p is p50:
mix_plate_well(well, keep_last_tip=True)
mixed_wells.add(well)
p.done_tip()
for well in plate.wells():
if well not in mixed_wells:
mix_plate_well(well)
def plateEverythingAndMix():
plateMasterMix()
platePerWellWater()
plateStrandA()
plateStrandBAndMix()
####################################################################################################################
# Off to the races
####################################################################################################################
diluteStrands()
createMasterMix()
plateEverythingAndMix()