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Protocol_BEST-Purification_OT2_V2.py
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#################################
### BEST Library Purification ###
#################################
## Author Jonas Greve Lauritsen
## Automatic preparation of covaris plates based on csv input
############################
## User Input
Col_Number = 3 # Number of columns with samples
#############################
#### Package loading ####
from opentrons import protocol_api
#### Meta Data ####
metadata = {
'protocolName': 'Purification of Library Build',
'apiLevel': '2.12',
'author': 'Jonas Lauritsen <[email protected]>',
'description': 'Automated purification of a BEST library build. The user inputs the number of columns for purification.'}
#### Protocol Script ####
def run(protocol: protocol_api.ProtocolContext):
#### LABWARE SETUP ####
## Smart labware
magnet_module = protocol.load_module('magnetic module',7)
## Work plates
Library_plate = magnet_module.load_labware('biorad_96_wellplate_200ul_pcr')
Purified_plate = protocol.load_labware('biorad_96_wellplate_200ul_pcr',1)
## Purification materials
Resevoir = protocol.load_labware('nest_12_reservoir_15ml',10)
Beads = Resevoir['A1']
Ethanol1 = Resevoir['A2']
Ethanol2 = Resevoir['A3']
EBT = Resevoir['A5']
Waste1 = Resevoir['A12'] # Beads supernatant
Waste2 = Resevoir['A11'] # 1st ethanol wash
Waste3 = Resevoir['A10'] # 2nd ethanol wast
## Tip racks (2x 10 µL, 2x 200 µl)
tiprack_10_1 = protocol.load_labware('opentrons_96_filtertiprack_10ul',5)
tiprack_200_1 = protocol.load_labware('opentrons_96_filtertiprack_200ul',4)
tiprack_200_2 = protocol.load_labware('opentrons_96_filtertiprack_200ul',11)
tiprack_200_3 = protocol.load_labware('opentrons_96_filtertiprack_200ul',8)
tiprack_200_4 = protocol.load_labware('opentrons_96_filtertiprack_200ul',9)
tiprack_200_5 = protocol.load_labware('opentrons_96_filtertiprack_200ul',6)
tiprack_200_6 = protocol.load_labware('opentrons_96_filtertiprack_200ul',3)
#### PIPETTE SETUP ####
## Loading pipettes
m200 = protocol.load_instrument('p300_multi_gen2', mount='left', tip_racks=([tiprack_200_1,tiprack_200_2,tiprack_200_3,tiprack_200_4,tiprack_200_5,tiprack_200_6]))
m20 = protocol.load_instrument('p20_multi_gen2', mount='right', tip_racks=([tiprack_10_1]))
#### Beads drying time (seconds) ####
## Different drying times - Sat from the last removal of the first column. Total drying time is estimated to 4 mins 55 seconds (23s per pipetting cycle)
BeadsTime = (295, 272, 249, 226, 203, 180, 157, 134, 111, 88, 65, 42)
BeadsTime = BeadsTime[(Col_Number-1)] # Selecting the relevant drying time
############################### Lab Work Protocol ###############################
## The instructions for the robot to execute.
protocol.comment("STATUS: Purification of BEST Library Build Begun")
protocol.set_rail_lights(True)
## Addition of Magnetic beads - pipetting to be tested (viscousity).
protocol.comment("STATUS: Beads Transfer Begun")
for i in range(Col_Number):
Column = i*8 #Gives the index of the first well in the column
m200.transfer(volume = 75, source = Beads, dest = Library_plate.wells()[Column], new_tip = 'always', trash = False, mix_before = (5,75), mix_after = (6,90), rate=0.5)
## 5 minutes incubation at room temperature
protocol.delay(minutes = 5)
## Engaging magnetic module. 5 mins wait for beads attraction
magnet_module.engage(height_from_base = 14)
protocol.delay(minutes = 5)
## Discarding supernatant - to be tested: pipette positioning. - flowrates to be tested.
protocol.comment("STATUS: Discarding Supernatant")
for i in range(Col_Number):
Column = i*8 #Gives the index of the first well in the column
m200.transfer(volume = 140, source = Library_plate.wells()[Column], dest = Waste1, new_tip = 'always', trash = False, rate=0.5) # Collection tray/reservoir for liquids??
## Double ethanol washing
protocol.comment("STATUS: Ethanol Wash Begun")
for k in range(2): # Double wash
## Setting up the wash variables
if k == 0:
Ethanol_Tips = tiprack_200_3
Ethanol = Ethanol1
Waste = Waste2
protocol.comment("STATUS: First Wash Begun")
if k == 1:
Ethanol_Tips = tiprack_200_4
Ethanol = Ethanol2
Waste = Waste3
protocol.comment("STATUS: Second Wash Begun")
## Adding Ethanol.
m200.pick_up_tip(Ethanol_Tips.wells_by_name()['A1']) # Using 1 set of tips for all rows
for i in range(Col_Number):
Column = i*8 # Gives the index for the first well in the column
#m200.pick_up_tip(Ethanol_Tips.wells()[Column])
m200.mix(repetitions = 2, volume = 200, location = Ethanol)
m200.aspirate(volume = 200, location = Ethanol,rate = 0.7)
m200.dispense(volume = 220, location = Library_plate.wells()[Column].top(1)) # Dispenses ethanol from 1 mm above the top of the well.
m200.blow_out(location = Waste) # Blow out to remove potential droplets
m200.touch_tip(location = Waste)
m200.return_tip()
## Removing Ethanol - reusing the tips from above (should eliminate droplet formation due to pre-wetting).
for i in range(Col_Number):
Column = i*8 # Gives the index for the first well in the column
m200.pick_up_tip(Ethanol_Tips.wells()[Column])
m200.aspirate(volume = 200, location = Library_plate.wells()[Column].bottom(z = 0.8),rate = 0.8)
m200.dispense(volume = 240, location = Waste)
m200.return_tip()
## Extra ethanol removal step to remove leftover ethanol before drying beads.
for i in range(Col_Number):
Column = i*8
m20.transfer(volume = 10, source = Library_plate.wells()[Column].bottom(z = 0.8), dest = Waste3, rate = 0.8, new_tip = 'always', trash = False)
## Drying beads (5 mins)
protocol.comment("STATUS: Drying Beads - time autoadjusted based on number of columns")
protocol.delay(seconds = BeadsTime) #Times to be verified given m20 step.
## Disengaging magnet
magnet_module.disengage()
## Adding EBT buffer.
protocol.comment("STATUS: EBT Buffer Transfer begun")
for i in range(Col_Number):
Column = i*8 #Gives the index for the first well in the column
m200.transfer(volume = 40, source = EBT, dest = Library_plate.wells()[Column], new_tip = 'always', trash = False, mix_after = (5,30))
## Incubation of library plate
protocol.pause('Seal library plate and spin it down shortly. Incubate the library plate for 10 min at 37*C. Press RESUME, when library plate has been returned (without seal) to the magnet module.')
## Engaging Magnet. 5 mins wait for beads withdrawal
magnet_module.engage(height_from_base = 14)
protocol.delay(minutes = 5)
## Transferring purified library to a new plate (purified plate). Transfer is sat higher to remove all.
protocol.comment("STATUS: Transfer of Purified Library")
for i in range(Col_Number):
Column = i*8 #Gives the index for the first well in the column
m200.transfer(volume = 50, source = Library_plate.wells()[Column], dest = Purified_plate.wells()[Column], new_tip = 'always', trash = False, rate = 1)
## Deactivating magnet module
magnet_module.disengage()
## Protocol finished
protocol.set_rail_lights(False)
protocol.comment("STATUS: Protocol Completed.")