## LOADING IN OPENTRONS SOFTWARE
from opentrons import types
import subprocess
metadata = {
'protocolName': 'Station B Zymo EUA',
'author': 'Chaz <[email protected]>, Dipro <[email protected]>',
'source': 'Covid-19 Diagnostics',
'apiLevel': '2.2'
}
## SPECIFYING PLATE TYPE WITH DIMENSIONS
plate_type = 'labcon'
# plate_type (above) should be 'labcon', 'nest', or 'ils'
## LOADING IN AUDIO
AUDIO_FILE_PATH = '/etc/audio/speaker-test.mp3'
def run_quiet_process(command):
subprocess.check_output('{} &> /dev/null'.format(command), shell=True)
def test_speaker():
print('Speaker')
print('Next\t--> CTRL-C')
try:
run_quiet_process('mpg123 {}'.format(AUDIO_FILE_PATH))
except KeyboardInterrupt:
pass
print()
## DEFINING THE ENTIRE PROTOCOL - THIS IS THE BULK OF THE PROTOCOL
def run(protocol):
# load labware and pipettes
sample_no = 48
samps = int(sample_no/8)
tips200 = [
protocol.load_labware(
'opentrons_96_tiprack_300ul', s) for s in [
'5', '6', '9', '7', '10'
]
]
all_tips = [tr['A'+str(i)] for tr in tips200 for i in range(1, 13)]
[tips1, tips2, tips3, tips4, tips5,
tips6, tips7, tips8, tips9, tips10] = [
all_tips[i:i+samps] for i in range(0, samps*10, samps)
]
p300 = protocol.load_instrument(
'p300_multi_gen2', 'left')
magdeck = protocol.load_module('magdeck', '4')
magheight = 13.7
plate_defs = {
'nest': 'nest_96_wellplate_2ml_deep',
'labcon': 'labcon_96_wellplate_2ml_deep',
'ils': 'ils_96_wellplate_2ml_deep'}
magplate = magdeck.load_labware(plate_defs[plate_type])
flatplate = protocol.load_labware('nest_96_wellplate_tempblock', '3')
liqwaste = protocol.load_labware(
'nest_1_reservoir_195ml', '11', 'Liquid Waste')
waste = liqwaste['A1'].top()
trough = protocol.load_labware(
'nest_12_reservoir_15ml', '2', 'Trough with Reagents')
trough_eth = protocol.load_labware(
'nest_12_reservoir_15ml', '1', 'Trough with Ethanol')
buffer = [trough[x] for x in ['A1', 'A2', 'A3'] for _ in range(2)][:samps]
wb1 = [trough[x] for x in ['A5', 'A6'] for _ in range(3)][:samps]
wb2 = [trough[x] for x in ['A8', 'A9'] for _ in range(3)][:samps]
ethanol1 = [trough_eth['A'+str(i)] for i in range(1, 7)]
ethanol2 = [trough_eth[x] for x in ['A7', 'A8'] for _ in range(3)]
water = trough['A12']
magsamps = [magplate['A'+str(i)] for i in range(1, 12, 2)][:samps]
elutes = [flatplate['A'+str(i)] for i in range(1, 7)][:samps]
p300.flow_rate.aspirate = 50
p300.flow_rate.dispense = 150
p300.flow_rate.blow_out = 300
magdeck.disengage()
## EVERYTHING BEFORE THIS LOADS IN THE PLATES, PIPETTES, TIPRACKS, and DEFINES WHERE REAGENTS WILL BE PLACED TO TELL
## THE ROBOT WHERE ITS PIPETTE IS IN SPACE AND WHAT IT'S HANDLING
## THIS CREATES A REPEPTITIVE FUNCTION FOR A WASH MIX THAT WILL BE USED LATER
# def wash mix - dispense on pellet
def wash_mix(reps, loc, vol):
loc1 = loc.bottom().move(types.Point(x=1, y=0, z=3))
loc2 = loc.bottom().move(types.Point(x=-1, y=0, z=3))
p300.aspirate(20, loc2)
mvol = vol-20
for _ in range(reps-1):
p300.aspirate(mvol, loc2)
p300.dispense(mvol, loc1)
p300.dispense(20, loc2)
## 800 µL OF BUFFER IS TRANSFERRED AT THIS STEP AND WILL ADD THE BUFFER & BEADS TO THE SAMPLES
protocol.comment('Adding viral buffer + beads to samples:')
for well, reagent, tip in zip(magsamps, buffer, tips1):
p300.pick_up_tip(tip)
for _ in range(3):
p300.aspirate(155, reagent)
p300.dispense(155, well.top(-5))
p300.aspirate(10, well.top(-5))
p300.aspirate(155, reagent)
p300.dispense(185, well.top(-10))
wash_mix(5, well, 180)
p300.blow_out()
p300.aspirate(20, well.top(-5))
p300.drop_tip()
## THE MAGNETIC BEADS WILL BE MIXED FOR 10 MINUTES HERE
protocol.comment('Mixing samples+buffer+beads:')
for well, tip, tret in zip(magsamps, tips2, tips1):
p300.pick_up_tip(tip)
wash_mix(15, well, 180)
p300.blow_out()
p300.aspirate(20, well.top(-5))
p300.drop_tip(tret)
## THERE WAS A 5 MINUTE INCUBATION STEP FOR THE OPENTRONS MAGNET -- THIS TIME WILL LIKELY BE ADJUSTED FOR THE BIOMEK
magdeck.engage(height=magheight)
protocol.comment('Incubating on magdeck for 5 minutes')
protocol.delay(minutes=5)
## THIS CREATES A REPETITIVE FUNCTION FOR REMOVING SUPERNATANT
def supernatant_removal(vol, src, dest, ht):
p300.flow_rate.aspirate = 20
tvol = vol
asp_ctr = 0
while tvol > 180:
p300.aspirate(
180, src.bottom().move(types.Point(x=-1, y=0, z=2)))
p300.dispense(180, dest)
p300.aspirate(10, dest)
tvol -= 180
asp_ctr += 1
p300.aspirate(
tvol, src.bottom().move(types.Point(x=-1, y=0, z=ht)))
dvol = 10*asp_ctr + tvol
p300.dispense(dvol, dest)
p300.flow_rate.aspirate = 50
## THIS EXECUTES REMOVING SUPERNATANT
for well, tip in zip(magsamps, tips1):
p300.pick_up_tip(tip)
supernatant_removal(540, well, waste, 2)
p300.aspirate(20, waste)
p300.drop_tip()
for well, tip in zip(magsamps, tips3):
p300.pick_up_tip(tip)
supernatant_removal(440, well, waste, 0.5)
p300.aspirate(20, waste)
p300.drop_tip()
magdeck.disengage()
## THIS CREATES A REPETITIVE FUNCTION THAT IS USED FOR WASH STEPS. THE ZYMO PROTOCOL FEATURES FOUR WASH STEPS:
## THE FIRST WASH STEP IS FOR WASH BUFFER 1
## THE SECOND WASH STEP IS FOR WASH BUFFER 2
## THE THIRD AND FOURTH WASH STEPS UTILIZE ETHANOL
## EACH WASH STEP CONTAINS A SMALL 3 MIN INCUBATION PERIOD WHEN ON THE MAGNET, FOLLOWED BY REMOVING SUPERNATANT FROM THE WASH
## THE OT2 ROBOT WAS HANDLING 500 µL OF WASH BUFFER 1, 500 µL OF WASH BUFFER 2, 900 µL OF ETHANOL WASH 1, 500 µL OF ETHANOL WASH 2.
def wash_step(src, vol, mtimes, tips, usedtips, msg, trash_tips=False):
protocol.comment(f'Wash Step {msg} - Adding to samples:')
for well, tip, tret, s in zip(magsamps, tips, usedtips, src):
p300.pick_up_tip(tip)
asp_ctr2 = 0
mvol = vol
while mvol > 180:
p300.aspirate(180, s)
p300.dispense(180, well.top(-3))
p300.aspirate(10, well.top(-3))
asp_ctr2 += 1
mvol -= 180
p300.aspirate(mvol, s)
dvol = 10*asp_ctr2 + mvol
p300.dispense(dvol, well.bottom(5))
wash_mix(mtimes, well, 180)
p300.blow_out()
p300.drop_tip(tret)
magdeck.engage(height=magheight)
protocol.comment('Incubating on MagDeck for 3 minutes.')
protocol.delay(minutes=3)
protocol.comment(f'Removing supernatant from Wash {msg}:')
svol = vol if vol == 900 else vol+40
for well, tip in zip(magsamps, usedtips):
p300.pick_up_tip(tip)
supernatant_removal(svol, well, waste, 0.5)
p300.aspirate(20, waste)
if trash_tips:
# p300.drop_tip()
p300.drop_tip(tip)
else:
p300.drop_tip(tip)
magdeck.disengage()
wash_step(wb1, 500, 20, tips4, tips2, '1 Wash Buffer 1')
wash_step(wb2, 500, 10, tips5, tips3, '2 Wash Buffer 2')
wash_step(ethanol1, 900, 10, tips6, tips4, '3 Ethanol 1')
wash_step(ethanol2, 500, 10, tips7, tips5, '4 Ethanol 2', trash_tips=True)
## THERE IS A STEP INCLUDED TO DRY ETHANOL. THIS IS BECAUSE ETHANOL IS A POWERFUL PCR INHIBITOR AND WE DON'T WANT THAT INCLUDED WITH THE SAMPLES.
protocol.comment('Allowing beads to air dry for 2 minutes.')
protocol.delay(minutes=2)
## THE ROBOT IS TOLD TO REMOVE EXCESS ETHANOLS FROM WELL
p300.flow_rate.aspirate = 20
protocol.comment('Removing any excess ethanol from wells:')
for well, tip, tret in zip(magsamps, tips8, tips6):
p300.pick_up_tip(tip)
p300.transfer(
180, well.bottom().move(types.Point(x=-0.5, y=0, z=0.4)),
waste, new_tip='never')
p300.drop_tip(tret)
p300.flow_rate.aspirate = 50
## THERE IS A 10 MIN INCUBATION PERIOD BEFORE ADDING NUCLEASE FREE WATER -- THIS IS DONE IN THE MANUAL PROTOCOL AS WELL
protocol.comment('Allowing beads to air dry for 10 minutes.')
protocol.delay(minutes=10)
magdeck.disengage()
## NUCLEASE FREE WATER IS ADDED TO THE WELLS TO PERFORM THE ELUTION
protocol.comment('Adding NF-Water to wells for elution:')
for well, tip, tret in zip(magsamps, tips9, tips7):
p300.pick_up_tip(tip)
p300.aspirate(20, water.top())
p300.aspirate(60, water)
# should we try 100 here
for _ in range(15):
p300.dispense(
50, well.bottom().move(types.Point(x=1, y=0, z=2)))
p300.aspirate(
50, well.bottom().move(types.Point(x=1, y=0, z=0.5)))
p300.dispense(50, well)
p300.dispense(30, well.bottom(6))
p300.aspirate(20, well.top(-3))
p300.drop_tip(tret)
## THERE IS AN INCUBATION PERIOD AT WHICH POINT STAFF CAN READY THE PCR PLATE FOR ELUTION
protocol.comment('Incubating at room temp for 10 minutes. Add PCR plate')
test_speaker()
protocol.delay(minutes=10)
## THE OT2 TRANSFERS THE ELUTES TO THE CLEAN 96-WELL PCR PLATE
magdeck.engage(height=magheight)
protocol.comment('Incubating on MagDeck for 2 minutes.')
protocol.delay(minutes=2)
protocol.comment('Transferring elution to final plate:')
p300.flow_rate.aspirate = 10
for src, dest, tip, tret in zip(magsamps, elutes, tips10, tips8):
p300.pick_up_tip(tip)
p300.aspirate(60, src.bottom().move(types.Point(x=-1.5, y=0, z=0.5)))
p300.dispense(60, dest)
p300.aspirate(10, dest.top())
p300.drop_tip(tret)
protocol.comment('Congratulations!')
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