Sep 25, 2023

Public workspaceOpentrons Pipeline: PCR Preparation

DOI
dx.doi.org/10.17504/protocols.io.rm7vzx6y8gx1/v1
  • 1Imperial College London;
  • 2Conservación Amazónica-ACCA;
  • 3Universidad Austral de Chile;
  • 4Universidad Nacional Mayor de San Marcos;
  • 5Field Projects International;
  • 6Washington University in Saint Louis;
  • 7San Diego Wildlife Alliance
Alexandra Sacco
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzx6y8gx1/v1
Protocol Citation: Jhakelin Reyes Vasquez, P. Sánchez-Vendizú, thalia.silvestre, Gideon Erkenswick, Alexandra Sacco, Mrinalini Watsa 2023. Opentrons Pipeline: PCR Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx6y8gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 22, 2023
Last Modified: September 25, 2023
Protocol Integer ID: 88226
Keywords: Opentrons, PCR, Mastermix, automated, insitulabs, OT2
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: 9772
Gordon and Betty Moore Foundation
Grant ID: 9776
Abstract
This protocol is a general automated pipeline to prepare mastermix at the plate level in a sterile way. The protocol volumes can be edited, as can sample numbers, to change the scale of the operation and optimise it to fit any end-point PCR.

This protocol was developed and optimised for the following:
  • Platform: Opentrons OT-2 automated pipetting robot
  • Tested with software version 6.3.1
  • ReagentGoTaq G2 Hot Start Green Master MixPromegaCatalog #M742B
  • Tips Used: 1 box and 1 column of 20uL filtered Opentrons Tips
  • Number of samples: 91 samples and 5 controls in a 96 well plate

Important: In an ideal situation, there would be two OT-2 robots involved in this process: the first to prepare sterile reagents and make and aliquot the mastermix, and the second to add the template. In the absence of this facility, we have developed a means of keeping each ingredient on the deck singly, rather than having all ingredients open on the deck at once. So please take note of the protocol pauses in built to replace each ingredient with the next. In this way, no two ingredients are open inside the OT2 hood at the same time.

Python File Version 2.2.


Guidelines
Step 1: Please note we use a particular Taq but you can alter this to suit your needs. You can also edit your PCR mix recipe by following the template in this protocol.

Step 5: Import the labware file BEFORE you import your protocol or it will give an error. This protocol has been validated against Opentrons software app version 6.3.1

Step 10: Take note of the two possible ways of loading gDNA. A skirted plate will clip into the deck, and a non-skirted plate or strip tubes will need support (and the labware definition file attached to this protocol).

Step 12: Note that we have specific wells in which we load controls, and for this you will need to remove the tips from the box so no template is added accidentally. We then load our controls later in a PCR hood.





Materials
Protocol materials
ReagentNuclease-Free Water-25mLPromegaCatalog #MC1191
Step 1
ReagentGoTaq G2 Hot Start Green Master MixPromegaCatalog #M742B
Abstract, Step 1
Reagent10% Bleach
Before starting, Step 14
ReagentDistilled Water
Before starting, Step 14
Reagent70% Alcohol
In Before starting and 2 steps
Safety warnings
Attention
Use PPE to handle all samples and liquids. Make sure to open ALL ingredients outside the OT2 deck, and place the plates in the deck only after any aerosols have dispersed.
Before start
Clean the OT2 deck and walls with:
Reagent10% Bleach 1 rinse
ReagentDistilled Water 1 rinse
Reagent70% Alcohol 2 rinses

Note
Avoid wetting electronic parts.

Materials and reagents
Materials and reagents
Reagents List
ReagentGoTaq G2 Hot Start Green Master MixPromegaCatalog #M742B
Primers Forward and Reverse Concentration10 micromolar (µM) or specific for each primer.
ReagentNuclease-Free Water-25mLPromegaCatalog #MC1191
Materials List
2 X Opentrons 20µL Filter Tips
1 X Opentrons Thermocycler Module - GEN2*
1 X Nest skirted PCR Plate
1 X 96-well Opentrons Aluminum Block
1 X P20 Gen 2 Multichannel Pipette
1 X P20 Gen 2 Single Pipette (optional)
1 X Nonskirted PCR plate (optional, see step 10)
2 X plate seals
1 X 0.2mL PCR strip tubes
1 x 1.5 mL microcentrifuge tube

Note
The thermocycler module is not a critical component of this protocol, but removing it would require the user to make some small modifications to the protocol and labware definitions indicating the location of the destination plate, and the plate type. You can prepare the plate and remove it and run it on any protocol.

Items to prepare in advance
Items to prepare in advance
Prepare your Master Mix following your specific protocol.

ABCDE
start [ ]desired [ ]Volume (uL) per reaction Per 96 reactions
GoTaq G2 Hot Start Green Master Mix 2X1X6.25600
Forward Primer Specific for each primerSpecific for each primerSpecific for each primer
Reverse Primer Specific for each primerSpecific for each primerSpecific for each primer
Nuclease-Free Water to reach 10.5
Mastermix volume per reaction10.5
Template2
Final volume12.5
An example of a mastermix recipe.

Dispense Master Mix in all 8 wells of a 0.2mL PCR strip tube equally.
Total volume per strip tube well = MasterMix total volume divided by 8
Import the lab ware file to your Opentrons app: Download denville_96_aluminumblock_200ul.jsondenville_96_aluminumblock_200ul.json25KB

Load python file to Opentrons app: Download Automated Opentrons Pipeline for PCR Preparation V2.2 ISL.pyAutomated Opentrons Pipeline for PCR Preparation V2.2 ISL.py3KB

Attach a P20 Gen 2 Multichannel Pipette to the right pipette mount.

(Optional) Attach a P20 Gen 2 Single Pipette to the left pipette mount. Note this is not used in this protocol, but should you want to do less than a multiple of 8, you could edit the script to use this pipette to load those samples, potentially.
Arrange the OT-2 deck as follows for 96 samples in a plate:

Slot 1: 96-well Aluminum Block with 200uL PCR strip tube with prepared mastermix


Note
Keep the Aluminum Block cold until the protocol starts.
This slot is used for placing the master mix, and then later the genomic DNA template in a Nest skirted PCR Plate


Slot 3: Opentrons 20µL Filter Tips
Slot 6: Opentrons 20µL Filter Tips
Slot 7: Opentrons Thermocycler Module - GEN2 with an empty Nest skirted PCR Plate

Placement of Labware and Tips in the OT2 Deck for transferring samples and Master Mix.
Calibrate the deck in the Opentrons app and follow instructions on the app.

OT2 PCR script definitions
OT2 PCR script definitions
Master Mix
PCR Master Mix

Position: Slot 1, 1 0.2mL PCR strip tube in 96-well Aluminum Block
Name in the Deck: MM
Labware name in the script protocol: MM_and_samples_plate
Variable name in the script: MMix
gDNA samples
Template DNA

Position: Slot 1, 96-well Aluminum Block with Nest skirted PCR Plate
Name in the Deck: gDNA cleaned samples
Labware name in the protocol: MM_and_samples_plate
Variable name in the script: template
PCR samples
Destination for template + Master Mix

Position: Slot 7, Opentrons Thermocycler Module - GEN2 with Nest skirted PCR Plate empty
Name in the Deck: PCR plate
Labware name in the script protocol: final_PCR_plate
Variable name in the script: pcr_samples
Protocol variables definition
The following variables can be edited within the python script in a text editor program to fit your specific PCR setup needs, by changing the "MM_vol" and "template_vol" definitions in the third line of the script.

The default protocol is written to use Amount2 µL of Template + Amount10.5 µL of Master Mix to reach Amount12.5 µL as final volume.


Standard protocol variable definitions:
"sample_number" indicates the number of samples that you will process: 96
"MM_vol" indicates the volume of Master Mix per well: 10.5
"template_vol" indicates the template volume added to each well: 2

Note
It is better if sample_number is a multiple of 8 given that the OT-2 uses a multichannel pipettes for transferring samples

OT2 Protocol: Transferring mastermix and samples
OT2 Protocol: Transferring mastermix and samples
Transferring Master Mix to final_PCR_plate

  • Place the PCR strip tube (already containing the MasterMix) in the first column of the 96-well Aluminum Block

Amount10.5 µL of Master Mix is transferred from the PCR strip tube in the aluminum block in Slot 1 to the PCR plate in the Opentrons Thermocycler Module - GEN2 in Slot 7

Note: Only the first column of tips in the Slot 6 rack is used for dispensing Master Mix to all columns of PCR plate.

  • Once this step is finished the robot will PAUSE. During this PROTOCOL PAUSE, carefully remove the PCR strip tube of MasterMix and now replace it with the plate of gDNA.


Note
You have two options here:

1. Either load your gDNA in a skirted NEST 100uL plate, which will clip into Slot 1, after you remove the aluminum block. If you do, you don't need the additional labware definition we have provided, and can simply edit the script to give the definition of the NEST plate (available on the

OR

2. Leave the aluminum block in, and add
a) a set of strip tubes (no caps) with gDNA, OR
b) a non-skirted plate with gDNA.

Both products linked above will fit the labware definition provided in this protocol.



Then, resume the run to continue with Step 11.
Transferring gDNA samples to final_PCR_plate

Amount2 µL of template are transferred from MM_and_samples_plate in Slot 1 to final_PCR_plate in Opentrons Thermocycler Module - GEN2

Tips in Slot 3 are used for this step

Note
IMPORTANT NOTE: Take out tips in Slot 3 for established positions for controls:
Negative controls: Positions G3, B10
Positive controls: Positions B3, G10
Sample control: D6

Note
Samples are mixed in this step before and after transferring.

Note: Once finished, cover the plate with a seal to move it into the PCR cabinet to add the controls.
Manually adding controls
Manually adding controls
Adding controls

Add the controls in a sterile PCR cabinet, in the following pre-determined spaces.


123456
A
B
Positive control
C
D
Sample control
E
F
G
Negative control
H
123456
A
B
Positive control
C
D
E
F
G
Negative control
H

The PCR plate is now ready to be loaded into a thermal cycler.
CLEANUP
CLEANUP
Clean the OT2 deck and walls with:
Reagent10% BleachContributed by users 1 rinse
ReagentDistilled WaterContributed by users 1 rinse
Reagent70% AlcoholContributed by users 2 rinses
Note
Avoid wetting any electronic parts.


Clean OT2 module with:
Reagent70% AlcoholContributed by users 2 rinses

Note
Avoid wetting electronic parts.

Air dry OT2 robot and modules.