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ot2-moclo-transformation-ecoli's Introduction

OT2 Modular Cloning (MoClo) and Transformation in E. coli Workflow

Material for OT2 Modular Cloning and Transformation in E. coli Workflow

This workflow enables end-to-end molecular cloning using the OT2 liquid handling robot by OpenTrons. This includes:

  • Performing modular cloning (Golden Gate) reactions.
  • Transforming plasmids into E. coli using heat shock.
  • Plating cell transformations onto rectangular agar plates.

All code for this project is freely distributed for academic and commercial uses under the MIT license.

General Installation

  1. Confirm that you have Python 3 installed.

  2. Download OT2 APP from Opentrons' website.

  3. Clone the GitHub repository to a local computer.

Modular Cloning, Cell Transformation, and Cell Plating

Getting Started

Users looking to implement the OT2 Modular Cloning and Transformation in E. coli workflow are encouraged to consult the instructions. If you are looking to contribute to this project, please raise an issue or pull request. Otherwise, feel free to reach out to rychen58.

Initial setup

  1. Prepare 1 CSV file (which can be produced in Excel) representing input DNA plate map. The input DNA plate maps may contain up to 96 DNA fragments and vectors, 8 rows by 12 columns, with each cell containing the name for DNA parts and empty wells left blank. The CSV file should represent 96-well microplate format plates of DNA parts at the desire concentration chossen by the users.

  2. Prepare 1 CSV file representing the combination list for combinations of parts to assemble, with each row representing one assembly. Each row should have N columns with the names of the parts to assemble (which must match the names in the plate maps of step 1).

Generating protocol

  1. Run the OT2-MoClo/moclo_transformation/moclo_transform_generator.py using Python (e.g. typing python3 moclo_transform_generator.py in the command line). Select the plate map, combinations list, and an output folder for the protocol when prompted.

  2. A protocol named moclo_transform_protocol.py should be saved in the output folder.

Authors

ot2-moclo-transformation-ecoli's People

Contributors

rychen58 avatar rkrishnasanka avatar

Stargazers

Thomas Duigou avatar Lucas Levassor avatar avatar Ryan avatar sangyoung park avatar W.R. Jackson avatar Benedict Carling avatar

Watchers

James Cloos avatar avatar Swapnil Bhatia avatar Prashant Vaidyanathan avatar avatar avatar

Forkers

benedict-carling priyankavsetty helloworld7-beep

ot2-moclo-transformation-ecoli's Issues

Ran out of p10 tips when using opentrons simulator

I used the example files and got the file moclo_transform_protocol.py as an output.

I then ran in with opentrons simulator and got the following error:
RuntimeWarning: p10_single has run out of tips

Below is the file I ran:

dna_plate_map_dict = {"input-dna-map": [["I13453_AB-1", "I13453_AB-2", "I13453_AB-4", "B0034m_BC-1", "C0012m_CD-1", "C0012m_CD-2", "C0012m_CD-4", "E0040m_C1N-1", "E0040m_C1N-2", "E0040m_C1N-4"], ["J23100_AB-1", "J23100_AB-2", "J23100_AB-4", "B0034m_BC-2", "C0040_CD-1", "C0040_CD-2", "C0040_CD-4", "3xFLAG_CC1-1", "3xFLAG_CC1-2", "3xFLAG_CC1-4"], ["J23102_AB-1", "J23102_AB-2", "J23102_AB-4", "B0034m_BC-4", "C0062_CD-1", "C0062_CD-2", "C0062_CD-4", "12 aa GS Linker_NO-1", "12 aa GS Linker_NO-2", "12 aa GS Linker_NO-4"], ["J23103_AB-1", "J23103_AB-2", "J23103_AB-4", "B0015_DE-1", "C0080_CD-1", "C0080_CD-2", "C0080_CD-4", "6xHIS_OD-1", "6xHIS_OD-2", "6xHIS_OD-4"], ["J23106_AB-1", "J23106_AB-2", "J23106_AB-4", "B0015_DE-2", "E0030_CD-1", "E0030_CD-2", "E0030_CD-4", "DVK_AE-1", "DVK_AE-2", "DVK_AE-4"], ["J23107_AB-1", "J23107_AB-2", "J23107_AB-4", "B0015_DE-4", "E0040m_CD-1", "E0040m_CD-2", "E0040m_CD-4", "DVK_CD-1", "DVK_CD-2", "DVK_CD-4"], ["J23116_AB-1", "J23116_AB-2", "J23116_AB-4", "", "E1010m_CD-1", "E1010m_CD-2", "E1010m_CD-4", "", "", ""], ["R0040_AB-1", "R0040_AB-2", "R0040_AB-4", "", "deGFP_CD-1", "deGFP_CD-2", "deGFP_CD-4", "", "", ""]]}

combinations_to_make = [{"name": "2-E0040m-1", "parts": ["E0040m_CD-1", "DVK_CD-1"]}, {"name": "2-C0012m-1", "parts": ["C0012m_CD-1", "DVK_CD-1"]}, {"name": "2-C0040-1", "parts": ["C0040_CD-1", "DVK_CD-1"]}, {"name": "2-C0062-1", "parts": ["C0062_CD-1", "DVK_CD-1"]}, {"name": "2-C0080-1", "parts": ["C0080_CD-1", "DVK_CD-1"]}, {"name": "2-E0030-1", "parts": ["E0030_CD-1", "DVK_CD-1"]}, {"name": "2-E1010m-1", "parts": ["E1010m_CD-1", "DVK_CD-1"]}, {"name": "2-deGFP-1", "parts": ["deGFP_CD-1", "DVK_CD-1"]}, {"name": "2-E0040m-2", "parts": ["E0040m_CD-2", "DVK_CD-2"]}, {"name": "2-C0012m-2", "parts": ["C0012m_CD-2", "DVK_CD-2"]}, {"name": "2-C0040-2", "parts": ["C0040_CD-2", "DVK_CD-2"]}, {"name": "2-C0062-2", "parts": ["C0062_CD-2", "DVK_CD-2"]}, {"name": "2-C0080-2", "parts": ["C0080_CD-2", "DVK_CD-2"]}, {"name": "2-E0030-2", "parts": ["E0030_CD-2", "DVK_CD-2"]}, {"name": "2-E1010m-2", "parts": ["E1010m_CD-2", "DVK_CD-2"]}, {"name": "2-deGFP-2", "parts": ["deGFP_CD-2", "DVK_CD-2"]}, {"name": "2-E0040m-4", "parts": ["E0040m_CD-4", "DVK_CD-4"]}, {"name": "2-C0012m-4", "parts": ["C0012m_CD-4", "DVK_CD-4"]}, {"name": "2-C0040-4", "parts": ["C0040_CD-4", "DVK_CD-4"]}, {"name": "2-C0062-4", "parts": ["C0062_CD-4", "DVK_CD-4"]}, {"name": "2-C0080-4", "parts": ["C0080_CD-4", "DVK_CD-4"]}, {"name": "2-E0030-4", "parts": ["E0030_CD-4", "DVK_CD-4"]}, {"name": "2-E1010m-4", "parts": ["E1010m_CD-4", "DVK_CD-4"]}, {"name": "2-deGFP-4", "parts": ["deGFP_CD-4", "DVK_CD-4"]}, {"name": "5-J23106-1", "parts": ["J23106_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-I13453-1", "parts": ["I13453_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-J23100-1", "parts": ["J23100_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-J23102-1", "parts": ["J23102_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-J23103-1", "parts": ["J23103_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-J23107-1", "parts": ["J23107_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-J23116-1", "parts": ["J23116_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-R0040-1", "parts": ["R0040_AB-1", "B0034m_BC-1", "E0040m_CD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "5-J23106-2", "parts": ["J23106_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-I13453-2", "parts": ["I13453_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-J23100-2", "parts": ["J23100_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-J23102-2", "parts": ["J23102_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-J23103-2", "parts": ["J23103_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-J23107-2", "parts": ["J23107_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-J23116-2", "parts": ["J23116_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-R0040-2", "parts": ["R0040_AB-2", "B0034m_BC-2", "E0040m_CD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "5-J23106-4", "parts": ["J23106_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-I13453-4", "parts": ["I13453_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-J23100-4", "parts": ["J23100_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-J23102-4", "parts": ["J23102_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-J23103-4", "parts": ["J23103_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-J23107-4", "parts": ["J23107_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-J23116-4", "parts": ["J23116_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "5-R0040-4", "parts": ["R0040_AB-4", "B0034m_BC-4", "E0040m_CD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8--J23106-1", "parts": ["J23106_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-I13453-1", "parts": ["I13453_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-J23100-1", "parts": ["J23100_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-J23102-1", "parts": ["J23102_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-J23103-1", "parts": ["J23103_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-J23107-1", "parts": ["J23107_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-J23116-1", "parts": ["J23116_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8-R0040-1", "parts": ["R0040_AB-1", "B0034m_BC-1", "3xFLAG_CC1-1", "E0040m_C1N-1", "12 aa GS Linker_NO-1", "6xHIS_OD-1", "B0015_DE-1", "DVK_AE-1"]}, {"name": "8--J23106-2", "parts": ["J23106_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-I13453-2", "parts": ["I13453_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-J23100-2", "parts": ["J23100_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-J23102-2", "parts": ["J23102_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-J23103-2", "parts": ["J23103_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-J23107-2", "parts": ["J23107_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-J23116-2", "parts": ["J23116_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8-R0040-2", "parts": ["R0040_AB-2", "B0034m_BC-2", "3xFLAG_CC1-2", "E0040m_C1N-2", "12 aa GS Linker_NO-2", "6xHIS_OD-2", "B0015_DE-2", "DVK_AE-2"]}, {"name": "8--J23106-4", "parts": ["J23106_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-I13453-4", "parts": ["I13453_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-J23100-4", "parts": ["J23100_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-J23102-4", "parts": ["J23102_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-J23103-4", "parts": ["J23103_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-J23107-4", "parts": ["J23107_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-J23116-4", "parts": ["J23116_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}, {"name": "8-R0040-4", "parts": ["R0040_AB-4", "B0034m_BC-4", "3xFLAG_CC1-4", "E0040m_C1N-4", "12 aa GS Linker_NO-4", "6xHIS_OD-4", "B0015_DE-4", "DVK_AE-4"]}]

#Modified version 2/28/20

'''
	Up to 88 rxns per single run and 24 rxns for triplicate of each combination of DNA assembly
	Multichannel p300, single channel p10
    This protocol is optimized for maximum walkaway time and percision
	'''

import time
import math

from opentrons import robot, instruments, labware, modules

'''
COLD_BLOCK = '96-PCR-tall-cold-block'
try:
	labware.create(
				   COLD_BLOCK,
				   grid=(12, 8),
				   spacing=(9, 9),
				   diameter=5,
				   depth=15.4,
				   volume=200)
except:
	print("Using existing labware definition for {0}".format(COLD_BLOCK))
'''

num_rxns = len(combinations_to_make)
#num_plates = math.ceil(num_rxns/24) # Amount of agar plates need for plating the transformed cells

'''
	For this protocol, the biorad_96_wellplate_200ul_pcr, is placed on the top of the TempDeck alone without the Opentrons 96 well aluminum block.
	'''

# Load in Bio-Rad 96 Well Plate on temp deck for moclos, transformation, and outgrowth.
temp_deck = modules.load('tempdeck', '10')
reaction_plate = labware.load('biorad_96_wellplate_200ul_pcr', '10', share=True)
temp_deck.set_temperature(10)

# Load in 1 10ul tiprack and 2 300ul tipracks
tr_10 = [labware.load('opentrons_96_tiprack_10ul', '3')]#, labware.load('opentrons_96_tiprack_10ul', '6')]
tr_300 = [labware.load('opentrons_96_tiprack_300ul', '6'), labware.load('opentrons_96_tiprack_300ul', '9')]
#for i in range(0, 1):
#   tr_300.append(labware.load('tipone_96_tiprack_200ul', '9'))

# Load in pipettes
p10_single = instruments.P10_Single(mount='right', tip_racks=tr_10)
p300_multi = instruments.P300_Multi(mount='left', tip_racks=tr_300)

''' Need to provide the instructions for loading reagent'''
reagents_plate = labware.load('biorad_96_wellplate_200ul_pcr', '4', 'Reagents Plate')
ligase = reagents_plate.wells('H12') #MoClo
restriction_enzyme = reagents_plate.wells('G12') #MoClo
buffer = reagents_plate.wells('F12') # MoClo

'''
	This deck slot location is dedicated for the reaction plate after MoClo protocol is completed, so at the beginning of the protocol there isn't an actual plate existing in this slot location.
	'''
post_moclo_reaction_plate = labware.load('biorad_96_wellplate_200ul_pcr', '7', 'Post-MoClo Reaction Plate')

# Load in water, SOC, and wash trough (USA Scientific 12 Well Reservoir 22ml)
trough = labware.load('usascientific_12_reservoir_22ml', '5', 'Reagents trough')
water = trough.wells(0) #Well 1
wash_0 = trough.wells(1) #Well 2
wash_1 = trough.wells(2) #Well 3
soc = trough.wells(3) #Well 4
liquid_waste = trough.wells(4) #Well 5

# Load in up to 2 DNA plates (Bio-Rad 96 Well Plate 200ul PCR)
#plate_name = dna_plate_map_dict.keys() # because there should be only 1 input plate
#input_dna_plate = labware.load('biorad_96_wellplate_200ul_pcr', '1', 'Input DNA Plate')
dna_plate_dict = {}
for plate_name in dna_plate_map_dict.keys():
	dna_plate_dict[plate_name] = labware.load('biorad_96_wellplate_200ul_pcr', '1', 'Input DNA Plate')

# Load in 1 agar plate, same antibiotic for all plasmids is assumed (e-gelgol)
# Be careful labware e-gelgol is an old container type, and could eventually be removed by Opentrons!
agar_plate = labware.load('e-gelgol', '2', 'Agar Plate')

#available_deck_slots = ['11', '8']

#____________________________________Start the MoClo protocol_______________________________

'''
	For this protocol, the biorad_96_wellplate_200ul_pcr, is placed on the top of the TempDeck alone without the metal part.
	'''
# Add water, buffer, restriction enzyme, ligase, and buffer to 2x master mix (2xMM).
#Prepare 2xMM for 2-part assembly at 20uL total volume
for i in range(2):
	p10_single.pick_up_tip()
	p10_single.transfer((150), water.bottom(), reaction_plate.wells(95-i).bottom(0.5), new_tip='never') #Water
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(24, buffer.bottom(), reaction_plate.wells(95-i).bottom(0.5), new_tip='never') #Buffer (1uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(95-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(6, ligase.bottom(), reaction_plate.wells(95-i).bottom(0.5), new_tip='never') #Ligase (0.25uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(95-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(12, restriction_enzyme.bottom(), reaction_plate.wells(95-i).bottom(0.5), new_tip='never') #Restriction Enzyme  (0.5uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(95-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()

#Prepare 2xMM for 5-part assembly at 20uL total volume
for i in range(2):
	p10_single.pick_up_tip()
	p10_single.transfer((78), water.bottom(), reaction_plate.wells(93-i).bottom(0.5), new_tip='never') #Water
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(24, buffer.bottom(), reaction_plate.wells(93-i).bottom(0.5), new_tip='never') #Buffer (1uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(93-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(6, ligase.bottom(), reaction_plate.wells(93-i).bottom(0.5), new_tip='never') #Ligase (0.25uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(93-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(12, restriction_enzyme.bottom(), reaction_plate.wells(93-i).bottom(0.5), new_tip='never') #Restriction Enzyme  (0.5uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(93-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()

#Prepare 2xMM for 8-part assembly at 20uL total volume
for i in range(2):
	p10_single.pick_up_tip()
	p10_single.transfer((6), water.bottom(), reaction_plate.wells(91-i).bottom(0.5), new_tip='never') #Water
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(24, buffer.bottom(), reaction_plate.wells(91-i).bottom(0.5), new_tip='never') #Buffer (1uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(91-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(6, ligase.bottom(), reaction_plate.wells(91-i).bottom(0.5), new_tip='never') #Ligase (0.25uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(91-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	p10_single.pick_up_tip()
	p10_single.transfer(12, restriction_enzyme.bottom(), reaction_plate.wells(91-i).bottom(0.5), new_tip='never') #Restriction Enzyme  (0.5uL/rxn)
	p10_single.mix(2, 10, reaction_plate.wells(91-i).bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()

# Add master mix to each wells that would contain rxn
# Add 2xMM to 2-part assembly
p10_single.pick_up_tip()
for i in range(12):
	p10_single.transfer(16, reaction_plate.wells(95).bottom(), reaction_plate.wells(i).bottom(0.3), new_tip='never')
	p10_single.blow_out()
p10_single.drop_tip()

p10_single.pick_up_tip()
for i in range(12):
	p10_single.transfer(16, reaction_plate.wells(94).bottom(), reaction_plate.wells(12+i).bottom(0.3), new_tip='never')
	p10_single.blow_out()
p10_single.drop_tip()

# Add 2xMM to 5-part assembly
p10_single.pick_up_tip()
for i in range(12):
	p10_single.transfer(10, reaction_plate.wells(93).bottom(), reaction_plate.wells(24+i).bottom(0.3), new_tip='never')
	p10_single.blow_out()
p10_single.drop_tip()

p10_single.pick_up_tip()
for i in range(12):
	p10_single.transfer(10, reaction_plate.wells(92).bottom(), reaction_plate.wells(36+i).bottom(0.3), new_tip='never')
	p10_single.blow_out()
p10_single.drop_tip()

# Add 2xMM to 8-part assembly
p10_single.pick_up_tip()
for i in range(12):
	p10_single.transfer(4, reaction_plate.wells(91).bottom(), reaction_plate.wells(48+i).bottom(0.3), new_tip='never')
	p10_single.blow_out()
p10_single.drop_tip()

p10_single.pick_up_tip()
for i in range(12):
	p10_single.transfer(4, reaction_plate.wells(90).bottom(), reaction_plate.wells(60+i).bottom(0.3), new_tip='never')
	p10_single.blow_out()
p10_single.drop_tip()

'''
	# Add 2xMM to 2-part assembly for varies volumes (5,10,20 uL)
	count = 0
	for i in range(3):
	for j in range(8):
	p10_single.pick_up_tip()
	p10_single.transfer(4*(2**i), reaction_plate.wells(89+(3*i)).bottom(0.3), reaction_plate.wells(3*j+count).bottom(0.3), new_tip='never')
	p10_single.blow_out()
	p10_single.drop_tip()
	count += 1
	
	# Add 2xMM to 5-part assembly for varies volumes (5,10,20 uL)
	count = 0
	for i in range(3):
	for j in range(8):
	p10_single.pick_up_tip()
	p10_single.transfer(2.5*(2**i), reaction_plate.wells(88+(3*i)).bottom(0.3), reaction_plate.wells(27+3*j+count).bottom(0.3), new_tip='never')
	p10_single.blow_out()
	p10_single.drop_tip()
	count += 1
	
	# Add 2xMM to 8-part assembly for varies volumes (5,10,20 uL)
	count = 0
	for i in range(3):
	for j in range(8):
	p10_single.pick_up_tip()
	p10_single.transfer(1*(2**i), reaction_plate.wells(87+(3*i)).bottom(0.3), reaction_plate.wells(54+3*j+count).bottom(0.3), new_tip='never')
	p10_single.blow_out()
	p10_single.drop_tip()
	count += 1
	'''

#This function checks the existance of DNA parts and returns for well location of the parts
def find_dna(name, dna_plate_map_dict, dna_plate_dict):
	"""Return a well containing the named DNA."""
	for plate_name, plate_map in dna_plate_map_dict.items():
		for i, row in enumerate(plate_map):
			for j, dna_name in enumerate(row):
				if dna_name == name:
					well_num = 8 * j + i
					return(dna_plate_dict[plate_name].wells(well_num))
	raise ValueError("Could not find dna piece named \"{0}\"".format(name))

#This function checks if the DNA parts exist in the DNA plates and returns for well locaion of output DNA combinations
def find_combination(name, combinations_to_make):
	"""Return a well containing the named combination."""
	for i, combination in enumerate(combinations_to_make):
		if combination["name"] == name:
			return reaction_plate.wells(i)
	raise ValueError("Could not find combination \"{0}\".".format(name))

combinations_by_part = {}
for i in combinations_to_make:
	name = i["name"]
	for j in i["parts"]:
		if j in combinations_by_part.keys():
			combinations_by_part[j].append(name)
		else:
			combinations_by_part[j] = [name]

#This section of the code combines and mix the DNA parts according to the combination list
for part, combinations in combinations_by_part.items():
	part_well = find_dna(part, dna_plate_map_dict, dna_plate_dict)
	combination_wells = [find_combination(x, combinations_to_make) for x in combinations]
	p10_single.pick_up_tip()
	while combination_wells:
		if len(combination_wells) > 5:
			current_wells = combination_wells[0:5]
			combination_wells = combination_wells[5:]
		else:
			current_wells = combination_wells
			combination_wells = []
		p10_single.aspirate(2 * len(current_wells), part_well.bottom(0.5))
		for i in current_wells:
			p10_single.dispense(2, i.bottom(0.5))
		if combination_wells:
			p10_single.mix(2, 10, wash_0.bottom(0.5))
			p10_single.blow_out()
			p10_single.mix(2, 10, wash_1.bottom(0.5))
			p10_single.blow_out()
	# Two washing steps are added to allow recycling of the tips
	p10_single.drop_tip()

# Incubate rxns for 2 hr (moclo), seal the Reaction Plate with adhesive film
'''
	Seal the Reaction Plate to avoid liquid evaporation.
	Discard the empty PCR tubes previously containing buffer, ligase, and restriction enzyme.
	Remove the Input_DNA_Plate from the Deck Space. Remaining DNA may be saved by sealing the Input_DNA_Plate with adhesive film and storing at -20°C
'''

'''start_time = time.time()
temp_deck.set_temperature(37)
p10_single.delay(minutes=120)
num_cols = math.ceil(num_rxns/8.0)

#Adding 4 ul of water halfway through.
	p10_single.pick_up_tip()
	for i in combinations_to_make:
	num_parts = len(i["parts"])
	# Add an extra 4 ul of water for evaporation.
	water_to_add = 4
	well = find_combination(i["name"], combinations_to_make)
	p10_single.transfer(water_to_add, water.bottom(), well.bottom(0.5), new_tip='never')
	p10_single.mix(4, 10, well.bottom(0.5))
	p10_single.mix(2, 10, wash_0.bottom(0.5))
	p10_single.blow_out()
	p10_single.mix(2, 10, wash_1.bottom(0.5))
	p10_single.blow_out()
	p10_single.drop_tip()
	time_elapsed = time.time() - start_time
	p10_single.delay(seconds=(120*60 - time_elapsed))

temp_deck.set_temperature(4)
'''
temp_deck.deactivate()
robot.pause()

# _____________________Start the Transformation protocol________________________________
'''
	The robotic liquid handler would automatically pause when the modular cloning protocol is completed, indicated by the blue light (cooling down) of the Temperature Module.
	Remove the reaction_plate from the top of the Temperature Module and place it on Slot 7 which is assigned to the post_moclo_reaction_plate at the beginning of the protocol, and remove the adhesive film.
	Before beginning the Cell Transformation protocol, load 10 μL/well of chemically competent cells into a new Bio-Rad 96 Well Plate and place it on the top of the Temperature Module.
	Be sure to un-pause the robot after completing all the steps listed above!
	'''

#for i in range(0, num_cols):
#Using letters for rows of custom container to maintain backwards compatibility.
#p300_multi.aspirate(10, reagents_plate.wells('A' + str(i + 1)).bottom(0.5))
#p300_multi.dispense(10, reaction_plate.wells(48 + i*8).bottom(0.5))
#p300_multi.drop_tip()

'''Use 10uL of competent cells with 2uL of each DNA rections from the MoClo protocol'''

# At the beginning of this for loop, the Bio-Rad 96 Well Plate (reaction_plate) now contains 10 uL of competent cells per wells (well 1 through 88)
p10_single.pick_up_tip()
# Add 2 ul of rxns to comp cells
for i in range(0, num_rxns):
	p10_single.transfer(2, post_moclo_reaction_plate.wells(i).bottom(0.5), reaction_plate.wells(i).bottom(0.5), new_tip='never')
	p10_single.mix(4, 10, reaction_plate.wells(i).bottom(0.5))
	p10_single.blow_out()
	p10_single.mix(2, 10, wash_0.bottom(0.5))
	p10_single.blow_out()
	p10_single.mix(2, 10, wash_1.bottom(0.5))
	p10_single.blow_out()
# Two washing steps are added to allow recycling of the tips
p10_single.drop_tip()

# Incubate at 4C, then heat shock.
'''Be sure to un-pause the robot in between the heat shock'''
temp_deck.set_temperature(4)
p10_single.delay(minutes=30)
temp_deck.set_temperature(42)
p10_single.delay(minutes=1)
temp_deck.set_temperature(4)
p10_single.delay(minutes=5)

# Add soc.
p300_multi.pick_up_tip()
for i in range(0, num_cols):
	p300_multi.transfer(150, soc.bottom(), reaction_plate.cols(i).bottom(1), new_tip='never')
	p300_multi.mix(2, 150, reaction_plate.cols(i).bottom(1))
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_0.bottom())
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_1.bottom())
	p300_multi.blow_out()
# Two washing steps are added to allow recycling of the tips
p300_multi.drop_tip()

# Grow for 1 hr, seal the plate with adhesive film to avoid evaporation
temp_deck.set_temperature(37)
p10_single.delay(minutes=60)
robot.pause()
temp_deck.deactivate()
'''
	Remove the adhesive film from the Reaction Plate before perceeding to Cell Plating.
	Be sure to un-pause the robot after removing the adhesive film!
	'''

# Dilute the recovered transformation reactions and start plating
'''All recovered transformation reactions are diluted to 10% of its original concentration before plating'''
#Dilution
p300_multi.pick_up_tip()
for i in range(0, num_cols):
	p300_multi.transfer(157, reaction_plate.cols(i).bottom(0.5), liquid_waste.bottom(), new_tip='never')
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_0.bottom())
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_1.bottom())
	p300_multi.blow_out()
	# Two washing steps are added to allow recycling of the tips
	p300_multi.transfer(45, soc.bottom(), reaction_plate.cols(i).bottom(0.5), new_tip='never')
	p300_multi.mix(2, 50, reaction_plate.cols(i).bottom(0.5))
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_0.bottom())
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_1.bottom())
	p300_multi.blow_out()
# Two washing steps are added to allow recycling of the tips
p300_multi.drop_tip()

#Plating
'''
	Un-comment line 328, and line 333 through line 339 if you're plating for triplicate of each reactions.
	'''
#count2 = 0
for i in range(0, num_cols):
	p300_multi.pick_up_tip()
	p300_multi.transfer(10, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i).bottom(0.3), new_tip='never')
	#p300_multi.transfer(1, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i).bottom(-1), new_tip='never')
	#p300_multi.transfer(9, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i+count2).bottom(0.8), new_tip='never')
	#p300_multi.transfer(1, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i+count2).bottom(-1), new_tip='never')
	#p300_multi.transfer(9, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i+count2+1).bottom(0.8), new_tip='never')
	#p300_multi.transfer(1, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i+count2+1).bottom(-1), new_tip='never')
	#p300_multi.transfer(9, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i+count2+2).bottom(0.8), new_tip='never')
	#p300_multi.transfer(1, reaction_plate.cols(i).bottom(0.5), agar_plate.cols(i+count2+2).bottom(-1), new_tip='never')
	#count2 += 2
	p300_multi.drop_tip()


'''
	# Dilute and plate.
	def spread_culture(source, dest, soc, dilute_after=True):
	p300_multi.mix(2, 150, source.bottom(0.5))
	p300_multi.aspirate(10, source.bottom(0.5))
	p300_multi.dispense(9, dest.top())
	p300_multi.dispense(1, dest.bottom(-1))
	if dilute_after:
	p300_multi.transfer(120, source.bottom(0.5), liquid_waste.bottom(), new_tip='never')
	p300_multi.mix(2, 300, wash_0.bottom())
	p300_multi.blow_out()
	p300_multi.mix(2, 300, wash_1.bottom())
	p300_multi.blow_out()
	p300_multi.transfer(120, soc, source.bottom(0.5), new_tip='never')
	
	for i in range(0, num_cols):
	agar_plate = agar_plates[i // 3]
	agar_well_num = (i % 3) * 8 * 4
	p300_multi.pick_up_tip()
	source = reaction_plate.wells(48 + i * 8)
	spread_culture(source, agar_plate.wells(agar_well_num), soc)
	spread_culture(source, agar_plate.wells(agar_well_num + 8), soc)
	spread_culture(source, agar_plate.wells(agar_well_num + 16), soc)
	spread_culture(source, agar_plate.wells(agar_well_num + 24), soc, dilute_after=False)
	p300_multi.drop_tip()
	'''

Any plans to update generated scripts for the v2 of the OT2 python API?

Hello,

Thanks for this great work. Is there any plans to update generated scripts for the v2 of the OT2 python API?

We are looking for scripts implementing Golden Gate assemblies with todays opentrons (present version of the opentron server is not anymore compatible with the v1 API).

Thanks,
Thomas

Request: Update for OT-2 v2 including the new thermocycler module

In the IWBDA presentation yesterday the benefits of using the new thermocycler module were discussed. Since this is only a feature of OT-2 v2, I was wondering if you have any plans for updating this repository to be compatible with OT-2 v2 and the thermocycler module.

Thank you!

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