Opentrons Pipeline: PCR Preparation
Thalía Silvestre, Jhakelin Reyes Vasquez, P. Sánchez-Vendizú, Gideon Erkenswick, Mrinalini Watsa, Alexandra Sacco
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
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- 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.
Before start
Clean the OT2 deck and walls with:
Steps
Materials and reagents
Reagents List
Primers Forward and Reverse 10micromolar (µM) or specific for each primer.
Materials List
2 X Opentrons 20µL Filter Tips
1 X Opentrons Thermocycler Module - GEN2*
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 1.5 mL microcentrifuge tube
Items to prepare in advance
Prepare your Master Mix following your specific protocol.
| A | B | C | D | E |
|---|---|---|---|---|
| start [ ] | desired [ ] | Volume (uL) per reaction | Per 96 reactions | |
| GoTaq G2 Hot Start Green Master Mix | 2X | 1X | 6.25 | =6.25*96 |
| Forward Primer | Specific for each primer | Specific for each primer | Specific for each primer | |
| Reverse Primer | Specific for each primer | Specific for each primer | Specific for each primer | |
| Nuclease-Free Water | to reach 10.5 | |||
| Mastermix volume per reaction | 10.5 | |||
| Template | 2 | |||
| Final volume | 12.5 |
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: denville_96_aluminumblock_200ul.json
Load python file to Opentrons app: Automated Opentrons Pipeline for PCR Preparation V2.2 ISL.py
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
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
Calibrate the deck in the Opentrons app and follow instructions on the app.
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 2µL of Template + 10.5µL of Master Mix to reach 12.5µL 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
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
10.5µL 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.
Then, resume the run to continue with Step 11.
Transferring gDNA samples to final_PCR_plate
2µL 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: Once finished, cover the plate with a seal to move it into the PCR cabinet to add the controls.
Manually adding controls
Adding controls
Add the controls in a sterile PCR cabinet, in the following pre-determined spaces.
The PCR plate is now ready to be loaded into a thermal cycler.
CLEANUP
Clean the OT2 deck and walls with:
Clean OT2 module with:
Air dry OT2 robot and modules.
