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assemble.py
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assemble.py
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import math
from modules.utils import provision_to_tube, printdatetime, det_new_group, thermocycle_ramp
from autoprotocol.pipette_tools import aspirate_source, depth
def assemble(protocol, params):
def provision_reagents(reagents, dest, num_rxts, mm_mult=1.3, num_rxts_plus=3.0):
for reagent in reagents.values():
protocol.provision(reagent['resource_id'], dest, "%s:microliter" % ((num_rxts + num_rxts_plus) * reagent['reagent_ratio'] * mm_mult))
def transfer_kwargs(pre_buffer, one_tip=False, one_source=False):
kwargs = {"one_tip": one_tip,
"one_source": one_source,
"pre_buffer": "%s:microliter" % pre_buffer,
"blowout_buffer": True}
return(kwargs)
# general parameters
ssDNA = params['ssDNA']
constructs = [construct['oligos'] for construct in params['constructs']]
num_constructs = len(constructs)
flattened = [val for oligo in constructs for val in oligo]
oligos = list({v: v for v in flattened}.values())
num_oligos = len(oligos)
mm_mult = 1.3
num_rxts_plus = 3
# provision water
water = protocol.ref( 'water', cont_type='96-deep', discard=True )
protocol.dispense_full_plate( water, 'water', '1000:microliter' )
# water = [provision_to_tube(protocol, "water%s" % (i + 1), "micro-2.0", "rs17gmh5wafm5p", 1900)
# for i in range(int(math.ceil(num_constructs/float(9.0))))
# ]
#
# for w in water:
# w.set_volume('1800:microliter')
# provision atp for entire protocol
atp_reagents = {'atp': {"resource_id": 'rs16pccshb6cb4',
'reagent_ratio': 0.1},
'water': {"resource_id": 'rs17gmh5wafm5p', 'reagent_ratio': 0.9}}
atp_rxts = (num_oligos + num_constructs)
atp = protocol.ref("atp_10mM", cont_type='micro-1.5', discard=True).well(0)
provision_reagents(atp_reagents, atp, atp_rxts, mm_mult, num_rxts_plus=6)
# provision kinase mix
kinase_mix = []
for i in range(int(math.ceil(num_oligos/60.0))):
kinase_mix.append(protocol.ref("kinase_mix-%s" % (i + 1), None, "micro-1.5", discard=True).well(0))
reagents = {'pnkbuffer': {"resource_id": 'rs16pc9rd5sg5d', "reagent_ratio": 3},
'water': {"resource_id": 'rs17gmh5wafm5p', "reagent_ratio": 18},
'pnk': {"resource_id": 'rs16pc9rd5hsf6', "reagent_ratio": 1}}
provision_reagents(reagents, kinase_mix, num_oligos, mm_mult, num_rxts_plus)
protocol.transfer(atp, kinase_mix, "%s:microliter" % ((num_oligos + num_rxts_plus) * 1 * mm_mult), new_group=True)
# kinase oligos
kinase_oligo_plate = protocol.ref("kinase_oligo_plate_%s" % printdatetime(time=False), None, "96-pcr",
discard=True)
wells_to_kinase = kinase_oligo_plate.wells_from("A1", num_oligos)
protocol.transfer(kinase_mix,
wells_to_kinase,
"23:microliter",
**transfer_kwargs(15, True, True))
for i, oligo in enumerate(oligos):
protocol.transfer(oligo,
wells_to_kinase[i],
"7:microliter",
mix_after=False,
new_group=det_new_group(i),
aspirate_source=aspirate_source(depth=depth("ll_following",
lld="pressure",
distance="0.0:meter")),
**transfer_kwargs(10))
protocol.seal(kinase_oligo_plate)
protocol.thermocycle(kinase_oligo_plate,
[{"cycles": 1, "steps": [
{"temperature": "37:celsius",
"duration": "60:minute"},
]}
], volume="30:microliter")
# dilute oligos
protocol.unseal(kinase_oligo_plate)
diluted_oligo_plate = protocol.ref("dilute_oligo_plate", None, "96-flat", discard=True)
diluted_oligo_wells = diluted_oligo_plate.wells_from(0, num_constructs)
protocol.transfer(water.all_wells(),
diluted_oligo_wells,
"200:microliter",
disposal_vol="0:microliter",
**transfer_kwargs(40, True, True))
for i, m in enumerate(constructs):
for kin_oligo in m:
index = list(oligos).index(kin_oligo)
protocol.transfer(kinase_oligo_plate.well(index), diluted_oligo_plate.well(i),
"2:microliter",
mix_after=False,
mix_vol="5:microliter")
diluted_oligo_plate.well(i).set_name(params['constructs'][i]['mutant_name'])
protocol.cover(diluted_oligo_plate)
protocol.spin(diluted_oligo_plate, "250:g", "2:minute")
# make ssDNA_mastermix
mm_mult_ssDNA = 1.5
mix_plate = protocol.ref("mix_plate", None, "96-pcr", discard=True)
ssDNA_mix = mix_plate.well(0)
protocol.transfer(ssDNA,
ssDNA_mix,
"%s:microliter" % ((num_constructs + num_rxts_plus) * 2.0 * mm_mult_ssDNA),
**transfer_kwargs((num_constructs + 1) * 1))
protocol.provision('rs17sh5rzz79ct', ssDNA_mix, "%s:microliter" % ((num_constructs + num_rxts_plus) * 0.2 * mm_mult_ssDNA))
# anneal oligos
protocol.uncover(diluted_oligo_plate)
annealing_plate = protocol.ref("annealing_oligo_plate", None, "384-pcr", storage="cold_20")
anneal_wells = annealing_plate.wells_from(0, num_constructs)
protocol.transfer(ssDNA_mix,
anneal_wells.wells,
"2.2:microliter",
dispense_speed="50:microliter/second",
**transfer_kwargs(7, True, True))
for dil_oligo, reaction in zip(diluted_oligo_wells.wells, anneal_wells.wells):
protocol.transfer(dil_oligo,
reaction,
"2:microliter",
aspirate_source=aspirate_source(depth("ll_bottom", distance=".001:meter")),
mix_after=True,
mix_vol="2:microliter",
flowrate="50:microliter/second",
repetitions=2,
new_group=det_new_group(i),
**transfer_kwargs(5))
reaction.set_name(dil_oligo.name)
protocol.seal(annealing_plate)
protocol.spin(annealing_plate, "250:g", "2:minute")
protocol.thermocycle(annealing_plate, [{
"cycles": 1,
"steps": thermocycle_ramp("95:celsius", "25:celsius", "60:minute", "4:minute")
}],
volume="5:microliter",
dataref=None,
dyes=None)
# polymerize
protocol.unseal(annealing_plate)
polymerize_MM = mix_plate.well(12)
reagents = {"buffer": {"resource_id": 'rs17sh5rzz79ct', "reagent_ratio": 0.6},
"t4ligase": {"resource_id": 'rs16pc8krr6ag7', "reagent_ratio": 0.4},
"t7polymerase": {"resource_id": 'rs16pca2urcz74', "reagent_ratio": 0.4},
"dntp": {"resource_id": 'rs16pcb542c5rd', "reagent_ratio": 0.4}
}
provision_reagents(reagents, polymerize_MM, num_constructs, mm_mult, num_rxts_plus)
protocol.transfer(atp, polymerize_MM, "%s:microliter" % ((num_constructs + num_rxts_plus) * 0.4 * mm_mult), new_group=True)
for reaction in anneal_wells.wells:
protocol.transfer(polymerize_MM,
reaction,
"2.2:microliter",
mix_after=False,
**transfer_kwargs(10))
if 'mutant_objs' in params.keys():
mut = next(m for m in params['mutant_objs'] if m.name == reaction.name)
mut.anneal_well = reaction
protocol.seal(annealing_plate)
protocol.incubate(annealing_plate, "ambient", "1.5:hour")
# pass plate back for unsealing
return annealing_plate
if __name__ == '__main__':
from autoprotocol.harness import run
run(assemble, "Assemble")