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| Original file line number | Diff line number | Diff line change |
|---|---|---|
| @@ -1 +1 @@ | ||
| """protocols.""" | ||
| """protocols.""" |
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| Original file line number | Diff line number | Diff line change |
|---|---|---|
| @@ -1,104 +1,114 @@ | ||
| """BMS PCR Protocol.""" | ||
|
|
||
| from opentrons.protocol_api import ParameterContext, ProtocolContext, Labware | ||
| from opentrons.protocol_api.module_contexts import ThermocyclerContext, TemperatureModuleContext | ||
| from opentrons.protocol_api.module_contexts import ( | ||
| ThermocyclerContext, | ||
| TemperatureModuleContext, | ||
| ) | ||
| from opentrons.protocol_api import SINGLE | ||
| from abr_testing.protocols import helpers | ||
| import threading | ||
| from opentrons.hardware_control.modules.types import ThermocyclerStep | ||
| from typing import List | ||
|
|
||
|
|
||
| metadata = { | ||
| 'protocolName': 'PCR Protocol with TC Auto Sealing Lid', | ||
| 'author': 'Rami Farawi <[email protected]', | ||
| } | ||
| requirements = { | ||
| 'robotType': 'OT-3', | ||
| 'apiLevel': '2.20' | ||
| "protocolName": "PCR Protocol with TC Auto Sealing Lid", | ||
| "author": "Rami Farawi <[email protected]", | ||
| } | ||
| requirements = {"robotType": "OT-3", "apiLevel": "2.20"} | ||
|
|
||
|
|
||
| def add_parameters(parameters: ParameterContext): | ||
| def add_parameters(parameters: ParameterContext) -> None: | ||
| """Parameters.""" | ||
| helpers.create_pipette_mount_parameter(parameters) | ||
| helpers.create_disposable_lid_parameter(parameters) | ||
| parameters.add_csv_file( | ||
| display_name = "Samples", | ||
| variable_name = "samples_csv", | ||
| description = "Asp/ disp volumes." | ||
| display_name="Samples", | ||
| variable_name="samples_csv", | ||
| description="Asp/ disp volumes.", | ||
| ) | ||
|
|
||
| def run(ctx: ProtocolContext)-> None: | ||
| """Protocol.""" | ||
| pipette_mount = ctx.params.pipette_mount | ||
| disposable_lid = ctx.params.disposable_lid | ||
| csv_samp = ctx.params.samples_csv | ||
|
|
||
| def run(ctx: ProtocolContext) -> None: | ||
| """Protocol.""" | ||
| pipette_mount = ctx.params.pipette_mount # type: ignore[attr-defined] | ||
| disposable_lid = ctx.params.disposable_lid # type: ignore[attr-defined] | ||
| parsed_csv = ctx.params.csv_data.parse_as_csv() # type: ignore[attr-defined] | ||
| rxn_vol = 50 | ||
| real_mode = True | ||
| # DECK SETUP AND LABWARE | ||
|
|
||
| tc_mod: ThermocyclerContext = ctx.load_module('thermocyclerModuleV2') | ||
| tc_mod: ThermocyclerContext = ctx.load_module( | ||
| "thermocyclerModuleV2" | ||
| ) # type: ignore[assignment] | ||
|
|
||
| tc_mod.open_lid() | ||
| tc_mod.set_lid_temperature(105) | ||
| temp_mod: TemperatureModuleContext = ctx.load_module('temperature module gen2', location='D3') | ||
| temp_mod: TemperatureModuleContext = ctx.load_module( | ||
| "temperature module gen2", location="D3" | ||
| ) # type: ignore[assignment] | ||
| reagent_rack = temp_mod.load_labware( | ||
| 'opentrons_24_aluminumblock_nest_1.5ml_snapcap') # check if 2mL | ||
| "opentrons_24_aluminumblock_nest_1.5ml_snapcap" | ||
| ) # check if 2mL | ||
|
|
||
| dest_plate = tc_mod.load_labware('opentrons_96_wellplate_200ul_pcr_full_skirt') # do I change this to tough plate if they run pcr? | ||
| dest_plate = tc_mod.load_labware( | ||
| "opentrons_96_wellplate_200ul_pcr_full_skirt" | ||
| ) # do I change this to tough plate if they run pcr? | ||
|
|
||
| source_plate = ctx.load_labware('opentrons_96_wellplate_200ul_pcr_full_skirt', location="D1") # do I change this to their plate? | ||
| source_plate = ctx.load_labware( | ||
| "opentrons_96_wellplate_200ul_pcr_full_skirt", location="D1" | ||
| ) # do I change this to their plate? | ||
|
|
||
| tiprack_50 = [ | ||
| ctx.load_labware("opentrons_flex_96_tiprack_50ul", slot) for slot in [8, 9] | ||
| ] | ||
|
|
||
| tiprack_50 = [ctx.load_labware('opentrons_flex_96_tiprack_50ul', slot) for slot in [8, 9]] | ||
|
|
||
| # Opentrons tough pcr auto sealing lids | ||
| if disposable_lid: | ||
| unused_lids = [ctx.load_labware("opentrons_tough_pcr_auto_sealing_lid", "C3")] | ||
| for i in range(2): | ||
| unused_lids.append(unused_lids[-1].load_labware("opentrons_tough_pcr_auto_sealing_lid")) | ||
| unused_lids.reverse() | ||
| used_lids = [] | ||
| unused_lids = helpers.load_disposable_lids(ctx, 3, ["C3"]) | ||
| used_lids: List[Labware] = [] | ||
|
|
||
| # LOAD PIPETTES | ||
| p50 = ctx.load_instrument( | ||
| "flex_8channel_50", pipette_mount, tip_racks=tiprack_50, liquid_presence_detection = True) | ||
| p50.configure_nozzle_layout( | ||
| style=SINGLE, | ||
| start="A1", | ||
| tip_racks=tiprack_50) | ||
| "flex_8channel_50", | ||
| pipette_mount, | ||
| tip_racks=tiprack_50, | ||
| liquid_presence_detection=True, | ||
| ) | ||
| p50.configure_nozzle_layout(style=SINGLE, start="A1", tip_racks=tiprack_50) | ||
| ctx.load_trash_bin("A3") | ||
| ctx.define_liquid(name="Mastermix", description='Mastermix', display_color='#008000') | ||
| ctx.define_liquid(name="Water", description='Water', display_color='#A52A2A') | ||
| ctx.define_liquid(name="DNA", description='DNA', display_color='#A52A2A') | ||
| mmx_liq = ctx.define_liquid( | ||
| name="Mastermix", description="Mastermix", display_color="#008000" | ||
| ) | ||
| water_liq = ctx.define_liquid( | ||
| name="Water", description="Water", display_color="#A52A2A" | ||
| ) | ||
| dna_liq = ctx.define_liquid(name="DNA", description="DNA", display_color="#A52A2A") | ||
|
|
||
| # mapping | ||
| csv_lines = [[val.strip() for val in line.split(',')] | ||
| for line in csv_samp.splitlines() | ||
| if line.split(',')[0].strip()][1:] | ||
|
|
||
|
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||
| temp_mod.set_temperature(4) | ||
|
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||
|
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| water = reagent_rack['B1'] | ||
| water = reagent_rack["B1"] | ||
| water.load_liquid(liquid=water_liq, volume=1500) | ||
| # | ||
| mmx_pic = reagent_rack.rows()[0] | ||
| for mmx_well in mmx_pic: | ||
| mmx_well.load_liquid(liquid=mmx_liq, volume=1500) | ||
|
|
||
| dna_pic = source_plate.wells() | ||
| for dna_well in dna_pic: | ||
| dna_well.load_liquid(liquid=dna_liq, volume=50) | ||
|
|
||
| # adding water | ||
| ctx.comment('\n\n----------ADDING WATER----------\n') | ||
| ctx.comment("\n\n----------ADDING WATER----------\n") | ||
| p50.pick_up_tip() | ||
| # p50.aspirate(40, water) # prewet | ||
| # p50.dispense(40, water) | ||
| for row in csv_lines: | ||
| num_of_rows = len(parsed_csv) | ||
| for row in range(num_of_rows): | ||
| water_vol = row[1] | ||
| if water_vol.lower() == 'x': | ||
| if water_vol.lower() == "x": | ||
| continue | ||
| water_vol = int(row[1]) | ||
| dest_well = row[0] | ||
|
|
@@ -111,14 +121,14 @@ def run(ctx: ProtocolContext)-> None: | |
| p50.configure_for_volume(50) | ||
|
|
||
| # p50.blow_out() | ||
| p50.drop_tip() | ||
| p50.drop_tip() | ||
|
|
||
| # adding Mastermix | ||
| ctx.comment('\n\n----------ADDING MASTERMIX----------\n') | ||
| ctx.comment("\n\n----------ADDING MASTERMIX----------\n") | ||
| for i, row in enumerate(csv_lines): | ||
| p50.pick_up_tip() | ||
| mmx_vol = row[3] | ||
| if mmx_vol.lower() == 'x': | ||
| if mmx_vol.lower() == "x": | ||
| continue | ||
|
|
||
| if i == 0: | ||
|
|
@@ -127,7 +137,7 @@ def run(ctx: ProtocolContext)-> None: | |
| mmx_tube = row[4] | ||
| if mmx_tube_check != mmx_tube: | ||
|
|
||
| p50.drop_tip() | ||
| p50.drop_tip() | ||
| p50.pick_up_tip() | ||
|
|
||
| if not p50.has_tip: | ||
|
|
@@ -148,50 +158,52 @@ def run(ctx: ProtocolContext)-> None: | |
| p50.drop_tip() | ||
|
|
||
| if p50.has_tip: | ||
| p50.drop_tip() | ||
| p50.drop_tip() | ||
|
|
||
| # adding DNA | ||
| ctx.comment('\n\n----------ADDING DNA----------\n') | ||
| ctx.comment("\n\n----------ADDING DNA----------\n") | ||
| for row in csv_lines: | ||
|
|
||
| dna_vol = row[2] | ||
| if dna_vol.lower() == 'x': | ||
| if dna_vol.lower() == "x": | ||
| continue | ||
|
|
||
| p50.pick_up_tip() | ||
|
|
||
| dna_vol = int(row[2]) | ||
| dest_and_source_well = row[0] | ||
|
|
||
|
|
||
| if dna_vol == 0: | ||
| break | ||
| p50.configure_for_volume(dna_vol) | ||
| p50.aspirate(dna_vol, source_plate[dest_and_source_well]) | ||
| p50.dispense(dna_vol, dest_plate[dest_and_source_well], rate=0.5) | ||
|
|
||
| p50.mix(10, 0.7*rxn_vol if 0.7*rxn_vol < 30 else 30, dest_plate[dest_and_source_well]) | ||
| p50.drop_tip() | ||
| p50.mix( | ||
| 10, | ||
| 0.7 * rxn_vol if 0.7 * rxn_vol < 30 else 30, | ||
| dest_plate[dest_and_source_well], | ||
| ) | ||
| p50.drop_tip() | ||
| p50.configure_for_volume(50) | ||
|
|
||
| ctx.comment('\n\n-----------Running PCR------------\n') | ||
| ctx.comment("\n\n-----------Running PCR------------\n") | ||
|
|
||
| if real_mode: | ||
|
|
||
| profile1: List[ThermocyclerStep] = [ | ||
| {'temperature': 95, 'hold_time_minutes': 2}, | ||
| {"temperature": 95, "hold_time_minutes": 2}, | ||
| ] | ||
| profile2: List[ThermocyclerStep] = [ | ||
|
|
||
| {'temperature': 98, 'hold_time_seconds': 10}, | ||
| {'temperature': 58, 'hold_time_seconds': 10}, | ||
| {'temperature': 72, 'hold_time_seconds': 30} | ||
| ] | ||
| profile3: List[ThermocyclerStep] = [ | ||
| {'temperature': 72, 'hold_time_minutes': 5} | ||
| {"temperature": 98, "hold_time_seconds": 10}, | ||
| {"temperature": 58, "hold_time_seconds": 10}, | ||
| {"temperature": 72, "hold_time_seconds": 30}, | ||
| ] | ||
| profile3: List[ThermocyclerStep] = [{"temperature": 72, "hold_time_minutes": 5}] | ||
| if disposable_lid: | ||
| lid_on_plate, unused_lids, used_lids = helpers.use_disposable_lid_with_tc(ctx, unused_lids, used_lids, dest_plate, tc_mod) | ||
| lid_on_plate, unused_lids, used_lids = helpers.use_disposable_lid_with_tc( | ||
| ctx, unused_lids, used_lids, dest_plate, tc_mod | ||
| ) | ||
| else: | ||
| tc_mod.close_lid() | ||
| tc_mod.execute_profile(steps=profile1, repetitions=1, block_max_volume=50) | ||
|
|
@@ -201,7 +213,7 @@ def run(ctx: ProtocolContext)-> None: | |
|
|
||
| tc_mod.open_lid() | ||
| if disposable_lid: | ||
| if len(used_lids) <= 1: | ||
| ctx.move_labware(lid_on_plate, "C2", use_gripper = True) | ||
| if len(used_lids) <= 1: | ||
| ctx.move_labware(lid_on_plate, "C2", use_gripper=True) | ||
| else: | ||
| ctx.move_labware(lid_on_plate, used_lids[-2], use_gripper = True) | ||
| ctx.move_labware(lid_on_plate, used_lids[-2], use_gripper=True) | ||
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