def get_values(*names): import json _all_values = json.loads("""{"num_samp":4,"reset_tipracks":true, "mix_reps2":5, "settling_1":4, "settling_3":3, "settling_drying":3, "wash1_vol":250,"wash2_vol":200, "wash3_vol":100, "elution_vol":60, "binding_buffer_vol":200,"final_vol":80,"heating_module_temp":60,"mag_height_1":3.1,"waste_water_mode":false,"asp_height":1,"length_from_side":2,"p300_mount":"left"}""") return [_all_values[n] for n in names] """Protocol.""" import os import csv from opentrons.types import Point metadata = { 'protocolName': 'RNA Extraction with Magnetic Life', 'author': 'Rami Farawi ', 'source': 'Custom Protocol Request', 'apiLevel': '2.11' } def run(ctx): """Protocol.""" [num_samp, reset_tipracks, mix_reps2, settling_1, settling_3, settling_drying, wash1_vol, wash2_vol, wash3_vol, elution_vol, binding_buffer_vol, final_vol, heating_module_temp, mag_height_1, waste_water_mode, asp_height, length_from_side, p300_mount] = get_values( # noqa: F821 "num_samp", "reset_tipracks", "mix_reps2", "settling_1", "settling_3","settling_drying", "wash1_vol", "wash2_vol", "wash3_vol", "elution_vol", "binding_buffer_vol", "final_vol", "heating_module_temp", "mag_height_1", "waste_water_mode", "asp_height", "length_from_side", "p300_mount") if not 0 <= num_samp <= 4: raise Exception('Please enter a sample number between 1-4') # load labware mag_mod = ctx.load_module('magnetic module gen2', '7') mag_plate = mag_mod.load_labware( 'innovationlaboratoryproducts_96_wellplate_1000ul', label='Mag Plate') reagent_tuberack = ctx.load_labware( 'opentrons_10_tuberack_falcon_4x50ml_6x15ml_conical', '4', label='Reagent Tuberack') waste_tuberack = ctx.load_labware( 'opentrons_6_tuberack_falcon_50ml_conical', '5', label='Waste Tuberack') tiprack300 = [ctx.load_labware('opentrons_96_tiprack_300ul', slot) for slot in ['8', '9']] temp_mod = ctx.load_module('temperature module gen2', '10') temp_rack = temp_mod.load_labware( 'opentrons_96_aluminumblock_biorad_wellplate_200ul', label='Temperature tuberack') tiprack200 = ctx.load_labware('opentrons_96_filtertiprack_200ul', '11') tuberack_lysis = ctx.load_labware('opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', '3') # load instrument p300 = ctx.load_instrument('p300_single_gen2', p300_mount, tip_racks=tiprack300) # Tip tracking between runs if not ctx.is_simulating(): file_path = '/data/csv/tiptracking.csv' file_dir = os.path.dirname(file_path) # check for file directory if not os.path.exists(file_dir): os.makedirs(file_dir) # check for file; if not there, create initial tip count tracking if not os.path.isfile(file_path): with open(file_path, 'w') as outfile: outfile.write("0, 0\n") tip_count_list = [] if ctx.is_simulating(): tip_count_list = [0, 0] elif reset_tipracks: tip_count_list = [0, 0] else: with open(file_path) as csv_file: csv_reader = csv.reader(csv_file, delimiter=',') tip_count_list = next(csv_reader) num_one = int(tip_count_list[0]) num_two = int(tip_count_list[1]) starting_tip_regular = num_one starting_tip_filter = num_two tips_300 = [tip for tiprack in tiprack300 for tip in tiprack.wells()] regular_tip_count = num_one regular_parked_tip_nums = {0: starting_tip_regular, 1: starting_tip_regular+1, 2: starting_tip_regular+2, 3: starting_tip_regular+3} def pick_up300(sample_tip=num_one, use_park=False): nonlocal regular_tip_count if regular_tip_count == 95: ctx.pause("Replace all 300ul non-filter tip racks") regular_tip_count = 0 p300.pick_up_tip(tips_300[regular_tip_count+num_samp]) regular_tip_count += 1 elif use_park: p300.pick_up_tip(tips_300[regular_parked_tip_nums[sample_tip]]) else: p300.pick_up_tip(tips_300[regular_tip_count+num_samp]) regular_tip_count += 1 filter_tip_count = num_two filter_parked_tip_nums = {0: starting_tip_filter, 1: starting_tip_filter+1, 2: starting_tip_filter+2, 3: starting_tip_filter+3} def pick_up_filter(sample_tip=num_two, use_park=False): nonlocal filter_tip_count if filter_tip_count == 95: ctx.pause("Replace all 200ul filter tip racks") filter_tip_count = 0 p300.pick_up_tip(tiprack200.wells()[filter_tip_count+num_samp]) filter_tip_count += 1 else: p300.pick_up_tip(tiprack200.wells()[filter_tip_count+num_samp]) filter_tip_count += 1 # load reagents lysis_buffer = reagent_tuberack.wells_by_name()['A2'].bottom(z=30) elution_buffer = reagent_tuberack.wells_by_name()['C2'].bottom(z=30) binding_buffer = reagent_tuberack.wells_by_name()['B2'].bottom(z=20) rps_wash_buffer = reagent_tuberack.wells_by_name()['A3'].bottom(z=20) wash_buffer = reagent_tuberack.wells_by_name()['A4'].bottom(z=20) calciumchloride_buffer = reagent_tuberack.wells_by_name()['B1'].bottom(z=20) samples_lysis = tuberack_lysis.columns()[0][:6:1][:num_samp] samples = mag_plate.columns()[0][:8:1][:num_samp] samples_second_well = mag_plate.columns()[1][:8:1][:num_samp] samples_third_well = mag_plate.columns()[2][:8:1][:num_samp] waste = waste_tuberack.wells()[0].bottom(z=80) def mixing(vol, index, loc, loc_length_from_side): ctx.comment('Mixing') side = 0 mix_loc3 = loc.bottom(z=5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.mix(10, vol, mix_loc3, rate = 10) def mixing_RPS(vol, index, loc, loc_length_from_side): ctx.comment('Mixing RPS') side = 1 mix_loc = loc.bottom(z=2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.mix(mix_reps2, vol, mix_loc, rate = 5) def mixing_RPS_1(vol, index, loc, loc_length_from_side): ctx.comment('Mixing RPS') side = 1 mix_loc_w = loc.bottom(z=3).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = 0 aspirate_loc = loc.bottom(z = 2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) for wash in range(15): p300.aspirate(vol, aspirate_loc, rate = 5) p300.dispense(vol, mix_loc_w, rate = 20) def mixing_lysis(vol, index, loc, loc_length_from_side): ctx.comment('Mixing RPS') side = 1 mix_loc_w = loc.bottom(z=4).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = 0 aspirate_loc = loc.bottom(z = 2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) for wash in range(30): p300.aspirate(vol, aspirate_loc, rate = 5) p300.dispense(vol, mix_loc_w, rate = 20) def aspirate_elution(vol, index, loc, loc_length_from_side): ctx.comment('Aspirate Elution') side = 0 aspirate_loc = loc.bottom(z = 3).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.aspirate(vol, aspirate_loc, rate = 5) def mixing_RPS_2(vol, index, loc, loc_length_from_side): ctx.comment('Mixing RPS') side = -1 mix_loc_w = loc.bottom(z=8).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = 0 aspirate_loc = loc.bottom(z = 2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) for wash in range(30): p300.aspirate(vol, aspirate_loc, rate = 5) p300.dispense(vol, mix_loc_w, rate = 20) def mixing_elution(vol, index, loc, loc_length_from_side): ctx.comment('Mixing Elution') side = -1 mix_loc_w = loc.bottom(z=8).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = 0 aspirate_loc = loc.bottom(z = 2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) for wash in range(30): p300.aspirate(vol, aspirate_loc, rate = 5) p300.dispense(vol, mix_loc_w, rate = 20) def remove_supernatant(vol, index, loc, loc_length_from_side): ctx.comment('Removing Supernatant') side = 0 dispense_loc = loc.bottom(z = 13.5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = -1 aspirate_loc = loc.bottom(z = asp_height).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) aspirate_bot = loc.bottom(z = 2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) aspirate_top = loc.bottom(z = 3).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) aspirate_mid = loc.bottom(z = 2.5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.dispense(0, dispense_loc) p300.aspirate(vol, aspirate_top, rate=0.1) p300.aspirate(vol, aspirate_mid, rate = 0.1) p300.aspirate(vol, aspirate_bot, rate=0.1) p300.dispense(vol*3, waste) def remove_beadbuffer(vol, index, loc, loc_length_from_side): ctx.comment('Removing Supernatant') side = 0 dispense_loc = loc.bottom(z = 13.5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = -1 aspirate_out = loc.bottom(z = 3).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.dispense(0, dispense_loc) p300.aspirate(vol, aspirate_out, rate=0.1) p300.dispense(vol, waste) def remove_supernatant_2(vol, index, loc, loc_length_from_side): ctx.comment('Removing Supernatant') side = 0 dispense_loc = loc.bottom(z = 13.5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = 1 aspirate_loc = loc.bottom(z = asp_height).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) aspirate_bot = loc.bottom(z = 2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) aspirate_top = loc.bottom(z = 3).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) aspirate_mid = loc.bottom(z = 3.5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.dispense(0, dispense_loc) p300.aspirate(vol, aspirate_top, rate=0.1) p300.aspirate(vol, aspirate_mid, rate = 0.1) p300.aspirate(vol, aspirate_bot, rate=0.1) p300.dispense(vol*3, waste) def dry_samp(vol, index, loc, loc_length_from_side): ctx.comment('dry sample') side = 0 dispense_loc = loc.bottom(z = 13.5).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) side = 1 aspirate_loc = loc.bottom(z = 0.2).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.dispense(0, dispense_loc) p300.aspirate(vol, aspirate_loc, rate=0.1) p300.dispense(vol, waste) p300.drop_tip() def dispense_height(vol, index, loc, loc_length_from_side): ctx.comment('dispense') side = 0 dispense_loc = loc.bottom(z = 10).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.dispense(vol, dispense_loc) def lysis_dispense(vol, index, loc, loc_length_from_side): ctx.comment('dispense') side = 0 dispense_loc = loc.bottom(z = 20).move( Point(x=(loc.diameter/2-loc_length_from_side)*side)) p300.dispense(vol, dispense_loc) # initialize temp mod and magnetic mod temp_mod.set_temperature(heating_module_temp) mag_mod.engage(height_from_base=mag_height_1) if not waste_water_mode: ctx.comment('\n\n\n\nNormal Mode') mag_mod.disengage() tips = [tip for rack in tiprack300 for tip in rack.wells()] tipcount = 0 def pick_up(): nonlocal tipcount p300.pick_up_tip(tips[tipcount]) tipcount += 1 mag_mod.engage(height_from_base=mag_height_1) # lysing samples ***************** for wash in range(1): for i, lysis in enumerate(tuberack_lysis.wells()[:num_samp]): pick_up() p300.aspirate(100, lysis_buffer) lysis_dispense(100, i, lysis, 2) p300.return_tip() tipcount = 0 for wash in range(1): for i, lysis in enumerate(tuberack_lysis.wells()[:num_samp]): pick_up() mixing_lysis(100, i, lysis, 2) p300.blow_out() p300.return_tip() ctx.delay(minutes = 3) tipcount = 0 for wash in range(3): for i, lysis in enumerate(tuberack_lysis.wells()[:num_samp]): pick_up() mixing_lysis(90, i, lysis, 2) p300.dispense(100, lysis) p300.blow_out() p300.return_tip() tipcount = 0 ctx.comment('End Normal Mode\n\n\n\n\n\n\n\n\n') # p300.reset_tipracks() ctx.delay(minutes=3) #Remove Storage Buffer From Beads for wash in range(1): for i, sample in enumerate(samples): pick_up() remove_beadbuffer(20, i, sample, 2) p300.return_tip() tipcount = 0 mag_mod.disengage() # Add CaCl2 and Move Lysed sample to mag Plate for wash in range(1): for i, (lysis, sample) in enumerate(zip(tuberack_lysis.wells()[:num_samp], samples)): pick_up300() p300.aspirate(100, calciumchloride_buffer) p300.dispense(100, lysis) mixing_lysis(150, i, lysis, 2) p300.drop_tip() ctx.delay(minutes = 3) for i, (lysis, sample) in enumerate(zip(tuberack_lysis.wells()[:num_samp], samples)): pick_up() mixing_RPS(150, i, lysis, 2) p300.aspirate(220, lysis) p300.dispense(220, sample) mixing_RPS(150, i, sample, 2) p300.return_tip() tipcount = 0 ctx.delay(minutes=7) # binding for wash in range(1): pick_up_filter() for i, sample in enumerate(samples): p300.aspirate(binding_buffer_vol, binding_buffer) dispense_height(binding_buffer_vol, i, sample, 2) p300.drop_tip() for i, sample in enumerate(samples): pick_up() mixing(300, i, sample, 1) p300.dispense(100, sample) p300.touch_tip() p300.drop_tip() mag_mod.engage(height_from_base=mag_height_1) ctx.delay(minutes=5) # remove supernatant for i, sample in enumerate(samples): pick_up300() dispense_height(0, i, sample, 2) remove_supernatant(100, i, sample, 1) remove_supernatant(50, i, sample, 1) p300.drop_tip() mag_mod.disengage() ctx.comment('End Normal Mode\n\n\n\n\n\n\n\n\n') if waste_water_mode: # remove storage buffer ctx.delay(minutes=settling_3) for i, sample in enumerate(samples): pick_up300(i, use_park=True) for _ in range(5): p300.aspirate(wash1_vol, sample) p300.dispense(wash1_vol, waste) p300.blow_out() p300.return_tip() mag_mod.disengage() ctx.comment('\n\n\n') #One RPS Wash and move ctx.comment('one wash') for wash in range(3): pick_up300() for i, sample in enumerate(samples): p300.aspirate(wash1_vol, rps_wash_buffer) dispense_height(wash1_vol, i, sample, 2) p300.drop_tip() for i, (sample, samp_well2) in enumerate(zip(samples, samples_second_well)): pick_up300() dispense_height(0, i, sample, 2) mixing_RPS_1(230, i, sample, 2) p300.aspirate(wash1_vol, sample) p300.dispense(wash1_vol, samp_well2) mixing_RPS_1(230, i, samp_well2, 2) p300.touch_tip() p300.drop_tip() mag_mod.engage(height_from_base=mag_height_1) ctx.delay(minutes=3) for i, samp_well2 in enumerate(samples_second_well): pick_up300() dispense_height(0, i, samp_well2, 2) remove_supernatant_2(100, i, samp_well2, 1) p300.drop_tip() mag_mod.disengage() #One Wash and moving sample to next clean well for wash in range(3): pick_up300() for i, samp_well2 in enumerate(samples_second_well): p300.aspirate(wash1_vol, wash_buffer) dispense_height(wash1_vol, i, samp_well2, 2) p300.drop_tip() for i, samp_well2 in enumerate(samples_second_well): pick_up300() dispense_height(0, i, samp_well2, 2) mixing_RPS_1(230, i, samp_well2, 2) p300.touch_tip() p300.drop_tip() mag_mod.engage(height_from_base=mag_height_1) ctx.delay(minutes=3) for i, samp_well2 in enumerate(samples_second_well): pick_up300() dispense_height(0, i, samp_well2, 2) remove_supernatant_2(100, i, samp_well2, 1) p300.drop_tip() mag_mod.disengage() #One Wash ctx.comment('One wash') for wash in range(2): pick_up300() for i, samp_well2 in enumerate(samples_second_well): p300.aspirate(wash1_vol, wash_buffer) dispense_height(wash1_vol, i, samp_well2, 2) p300.drop_tip() for i, samp_well2 in enumerate(samples_second_well): pick_up300() dispense_height(0, i, samp_well2, 2) mixing_RPS_2(230, i, samp_well2, 2) p300.touch_tip() p300.drop_tip() mag_mod.engage(height_from_base=mag_height_1) ctx.delay(minutes=3) for i, samp_well2 in enumerate(samples_second_well): pick_up300() dispense_height(0, i, samp_well2, 2) remove_supernatant_2(100, i, samp_well2, 1) p300.drop_tip() mag_mod.disengage() ctx.delay(minutes=settling_drying) #drying sample for i, samp_well2 in enumerate(samples_second_well): pick_up_filter() dispense_height(0, i, samp_well2, 2) dry_samp(50, i, samp_well2, 1) ctx.delay(minutes=settling_3) mag_mod.disengage() #Transfer for wash in range(1): pick_up_filter() for samp_well2, source in zip(samples_second_well, temp_rack.rows()[0][:12:1]): p300.aspirate(elution_vol, elution_buffer) dispense_height(elution_vol, i, samp_well2, 2) p300.drop_tip() for samp_well2, source in zip(samples_second_well, temp_rack.rows()[0][:12:1]): pick_up_filter(i, use_park = True) dispense_height(0, i, samp_well2, 2) mixing_elution(50, i, samp_well2, 1) p300.aspirate(200, samp_well2) dispense_height(200, i, source, 2) p300.aspirate(50, samp_well2) dispense_height(50, i, source, 2) p300.blow_out() p300.return_tip() ctx.delay(minutes=2) ctx.comment('\n\n\n') # elution on Aluminum block for i, (source, samp_well3) in enumerate(zip(temp_rack.rows()[0][:12:1], samples_third_well)): pick_up_filter(i, use_park=True) mixing(elution_vol, i, source, 2) p300.return_tip() ctx.delay(minutes=2) for i, (source, samp_well3) in enumerate(zip(temp_rack.rows()[0][:12:1], samples_third_well)): pick_up_filter(i, use_park=True) mixing(100, i, source, 2) aspirate_elution(200, i, source, 2) p300.dispense(200, samp_well3) p300.drop_tip() ctx.comment('\n\n\n') # Transfer mag_mod.engage(height_from_base=mag_height_1) ctx.delay(minutes=2) ctx.comment('\n\n\n') ctx.comment('\n\n\n') num_one = regular_tip_count num_two = filter_tip_count # write updated tipcount to CSV new_tip_count = str(num_one)+", "+str(num_two)+"\n" if not ctx.is_simulating(): with open(file_path, 'w') as outfile: outfile.write(new_tip_count)