TITLE: Opentrons  Pipeline: PCR Preparation
AUTHORS: Jhakelin Reyes Vasquez, P. Sánchez-Vendizú, thalia.silvestre, Gideon Erkenswick, Alexandra Sacco, Mrinalini Watsa

[DESCRIPTION]
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
 
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: 
  1 rinse 
   1 rinse 
  2 rinses

 


[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.






[STEPS]
SECTION: Materials and reagents
1. Reagents List
  
Primers Forward and Reverse 10 micromolar (µM) or specific for each primer.
  
SECTION: Materials and reagents
2. 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

 

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

      inventory IDstart conc.desired conc.Reaction number18.5Go Taq G2 Hot Start Green Master Mix216.2553.125forward P (10um)100.50.6255.3125reverse P (10um)100.50.6255.3125H2O (ultra pure)325.5 Volume:10.5

SECTION: Items to prepare in advance
5. Import the lab ware file to your Opentrons app:   

Load python file to Opentrons app:   

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. 
SECTION: Items to prepare in advance
5.1. 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 

 
SECTION: Items to prepare in advance
5.2. Calibrate the deck in the Opentrons app and follow instructions on the app. 

SECTION: OT2 PCR script definitions
7. 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
SECTION: OT2 PCR script definitions
6. 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
SECTION: OT2 PCR script definitions
8. 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
SECTION: OT2 PCR script definitions
9. 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 2 µL  of Template + 10.5 µL of Master Mix to reach 12.5 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  

 

SECTION: OT2 Protocol: Transferring mastermix and samples
10. 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 

10.5 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. 


 


Then, resume the run to continue with Step 11.
SECTION: Manually adding controls
12. Adding controls

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


 

SECTION: Manually adding controls
13. The PCR plate is now ready to be loaded into a thermal cycler. 
SECTION: Items to prepare in advance
4. 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
SECTION: OT2 Protocol: Transferring mastermix and samples
11. Transferring gDNA samples to final_PCR_plate

2 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: Once finished, cover the plate with a seal to move it into the PCR cabinet to add the controls.
SECTION: CLEANUP
14. Clean the OT2 deck and walls with: 
  1 rinse 
   1 rinse 
  2 rinses
 


SECTION: CLEANUP
15. Clean OT2 module with:
  2 rinses

 

SECTION: CLEANUP
16. Air dry OT2 robot and modules.