Oct 20, 2025
Version 3
OT-2 PCR sample preparation protocol V.3
- Ana Mariya Anhel1,
- Lorea Alejaldre1,
- Manuel Gimenez1,
- Ángel oñi-Moreno2
- 1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain;
- 2Centro Nacional de Biotecnología (CNB-CSIC)
- Ángel oñi-Moreno: [email protected];
Protocol Citation: Ana Mariya Anhel, Lorea Alejaldre, Manuel Gimenez, Ángel oñi-Moreno 2025. OT-2 PCR sample preparation protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpyznl5b/v3Version created by Lorea Alejaldre
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
We use this protocol and it's working
Created: June 07, 2024
Last Modified: October 20, 2025
Protocol Integer ID: 101396
Keywords: PCR sample preparation, automation, opentrons, pcr sample preparation protocol, pcr mastermix, pcr, spurious pcr sample, description of the lap repository entry lap, throughput workflow for the genotypic characterization, transposon insertion library variant, lap repository, protocol in the lap entry link, lap repository entry lap, version of the protocol, ot2, set of instruction, lap format script, protocol, protocol setup
Funders Acknowledgements:
Comunidad de Madrid
Grant ID: Y2020/TCS-6555, 2019-T1/BIO-14053
MCIN/AEI
Grant ID: CEX2020-000999-S, PID2020-117205GA-I00, CNS2022-135951, PID2023-152470NB-I00
European Research Council
Grant ID: 101044360
Abstract
This protocol is meant to create a PCR mastermix with 1 or more primer sets, distribute it in 1 or more 96-well plates, inoculate from 1 or more 96-well plates containing the template and run the reaction in an OT-2 thermocycler (if available).
This protocol is run by using a LAP format script and its corresponding .xlsx file were different customizable variables such as volumes of transfer, type of plates, number of primers per set, and availability of OT-2 thermocycler are indicated.
Specifically, this protocol provides a set of instructions or description of the LAP repository entry LAP-PCR-OT2-2.0.0. You can find the script and complementary information for this specific version of the protocol in the LAP entry link and GitHub link to LAP entry documents
In our laboratory, this protocol has been used as part of the "High-throughput workflow for the genotypic characterization of transposon insertion library variants" also available in protocols.io to prepare arbitrary and spurious PCR samples.
The current version incorporates the following modifications:
- Description of robot and protocol setup in a separate protocols.io entry (Setting and Customizing OT-2 for LAP Entries)
- When transferring the polymerase and primers, change tip every time it aspirates from source tubes
- Wells with controls can be anywhere in the plate
- New variable in Sheet 'ModuleVariables' called 'Max Volume Per Mix Tube In Shaker'
Guidelines
This protocol has been tested with cultures from Pseudomonas putida KT2440 and PCR products as part of the High-throughput workflow for the genotypic characterization of transposon insertion library variants. Software requirements to run this protocol are the following:
- Python 3 (Developed with Python 3.7.1)
- Opentrons Protocol API version >= 2.14
- Opentrons App software Version >= 7.0.2
- Python packages needed (version moment of development): opentrons (7.0.2), pandas (0.25.3), numpy (1.15.1), openpyxl (3.1.2), math, random
Materials
Software
- python 3.7.1
- python packages: pandas, random, math, numpy, opentrons, openpyxl
- OT App
- Excel
OT-2 Labware
- Opentrons Tipracks
Equipment
Opentrons 96 Tip Rack 300 µL
NAME
Tip rack
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Opentrons 96 Tip Rack 20 µL
NAME
Tip rack
TYPE
Opentrons
BRAND
-
SKU
LINK
- 96-well plates
Equipment
96-well plates, flat bottom, non treated
NAME
Cell culture plates
TYPE
VWR
BRAND
734-2781
SKU
LINK
- PCR plates
- Opentrons Falcon tuberack
Equipment
Opentrons 15 Tube Rack with Falcon 15 mL Conical
NAME
OT Tube Rack
TYPE
Opentrons
BRAND
-
SKU
LINK
- 1.5mL eppendorfs (without the cap)
- 4ºC cold-block with adaptor (file attach)
Equipment
BRAND™ Centrifuge Tube Mini-Cooler
NAME
ColdBlock
TYPE
BRAND
BRAND
10141921
SKU
LINK
adaptor_OT_coldblock.stl Reactives:
Phire Green Hot Start II PCR Master MixFisher ScientificCatalog #15391732 This is the reason why the transfering of the polymerase is done with a slower aspiration and dispensing rate, due to the viscosity of the reactive
Thermo Scientific™ Phire Hot Start II PCR Master MixFisher ScientificCatalog #15361732
MilliQ water
Oligonucleotides:
- For spurious integration control: PS3, PS4, PS5, PS6 (Silva-Rocha et al. 2013 doi: 10.1093/nar/gks1119)
- For arbitrary PCR: ARB2, ME-O-Km-Ext-F, ME-O-Km-Int-F, ME-O-Sm-Ext-F, ME-O-Sm-Int-F, ME-O-Gm-Ext-F, ME-O-Gm-Ext-R (Martinez-Garcia et al. 2013 https://doi.org/10.3389/fbioe.2014.00046)
- For optional insert control: PSMCS
Equipment:
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Equipment
Single Channel Electronic Pipette (GEN2) 300uL
NAME
Opentrons Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Single Channel Electronic Pipette (GEN2) 20uL
NAME
Opentrons Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Before start
Note that if more than one primer set wants to be used in the same final plate, the protocol should be re-run to create the new PCR master mix, the new starting position in the final plate should be specified.
Files Preparation
Preparing Customized Template
Preparing the template (a .xlsx) with the specific variables for each experiment.
Here we attach one Excel with several sheets and a PDF file explaining each variable:
- GeneralVariables: variables related mainly to the labware that is going to be used
- SamplesPlateVariables: variables related to the specifications of each source plate
- PipetteVariables: variables related to the pipette(s) that are going to be used
- ReagentsPerReaction: variables that will determine the final mix of the wells
- ModuleVariables: variables related to the modules used in the protocol, the thermocycler and the heater-shaker
- TemperatureProfile (Optional): PCR program that will be performed in the thermocycler if set as True in the ModuleVariables sheet
- Maps (Optional): sheet(s) with the names of the samples in the source plates and will be reflected in the final plate map(s) –> not included in the template but needed to be included if the variable Map IDs has a value.
PCRInstructions_v200.pdf 
TemplateVariablesPCR.xlsx Fill the template with the corresponding values (PCR conditions, number of primer pairs, output map, availability of OT-2 thermocycler etc...) and save file as VariablesPCR.xlsx (if the file has another name the LAP format script will not read the file).
Safety information
Verify volume range of the OT-2 pipettes available with the volume of a single reaction.
This can be a source of the errors displayed by the app in later steps
Store it with the name VariablesPCR.xlsx
Note
The file should be spelt exactly VariablesPCR.xlsx or the Python script won't work correctly
Transferring variables file to Robot
For this protocol to work we need to transfer the VariablesPCRs.xlsx to the directory /data/user_storage of the OT system that we will use to perform the protocol
As well, if we are using custom labware we need to upload it to the OT App and send it to the directory /data/labware/v2/custom_definitions/custom_beta if the labware is not there yet.
Finally, we need to make sure the package openpyxl is installed in the robot system
We can do this entire step by following the protocol Setting and Customizing OT-2 for LAP Entries with the specifications given in the text above
Running Protocol
Load script in OT-App
Now that we have transferred the variable files to the robot, we can load the script and run it in the selected robot
Note
This whole step has been developed and tested with version 7.0.2 of the OT-App
Indications may vary from version to version of the opentrons App and the version of the script.
Software
Opentrons App
NAME
Windows >=10, Mac >=10 , Ubuntu >=12.04
OS
Opentrons
DEVELOPER
REPOSITORY
Note
Updated script versions can be found at our Github
https://github.com/BiocomputationLab/LAPrepository/tree/main/LAPEntries or LAP repository https://www.laprepo.com/repository/ in the directory and entry named LAP-PCR-OT2 followed by the version.
Load the script in the App
Protocols -> Import -> Drag Python script
This version of the protocol was developed when the last version available of LAP-PCR-OT2 was the 2.0.0 which script you can find attached
ScriptPCR_v200.py Note
The App with version 7.0.2 analyzes your protocol before setting a robot to run, so the labware will not be shown before assigning the protocol to a specific robot when you import it into the App.
Note
The App with version 7.0.2 analyzes your protocol before setting a robot to run, so the labware will not be shown before assigning the protocol to a specific robot when you import it into the App.
Select Robot to Perform Script
Click in the protocol -> Start setup -> Choose the OT where the file VariablesPlateIncubation.xlsx is -> Proceed To Setup
After clicking on Proceed to Setup, you should obtain the labware positions in the Labware tab and the reagents, with their corresponding volume, in the Liquids tab.
In case the protocol with the set variables cannot run, an error will occur during the run. Many errors are contemplated already and have a specific message that hints the user what could have gone wrong.
Note
The volume of the initial samples is established to be 90% of the max volume of the well, but this is only a recommendation. Just make sure that there is enough volume to transfer to all the final plates.
On the other hand, the volume of the reagents is precisely what is needed, so it is suggested always to add more to consider the pipetting error.
Note
It is recommended that you perform a labware position check.
You can do it with test plates after loading the script but before cleaning the surface. That way, you reduce the probability of contamination (using test plates and labware) and pipetting errors (position check).
Run Protocol in OT
Make sure the needed calibrations are done
Labware position check is performed (if needed)
Wipe the surface of the deck with 70% ethanol to clean and desinfect the surfaces
Note
For more information in how to clean the robot go to the following link https://support.opentrons.com/s/article/Cleaning-the-robot-s-surfaces
Set the respective labwares in the slots as the OT App and the instruction file set
Safety information
Make sure that the source and final A1 wells are at the left top (common mistake), and each labware fits appropierly.
Set the different reactives in their respective coldblock positions, as stated in the instruction file
Safety information
Make sure that the caps of these 1.5mL eppendorfors are removed (cut them) so they are not hanging and the pipette(s) do not have the possibility to crash with them
Start Run
The procedure that the robot is going to do is mainly divided in 3 parts:
- Creation of primer sets combining water, primer(s) and polymerase
- Mixing and distributing the primer set(s)
- Transferring samples to the corresponding wells of all set of primers
Expected result
One or more final plates (usually PCR plates) with the mix for each set of primers combined with the colony samples and an excel file containig th emapping of each final plate in the folder /data/user_storage of the robot
After-running
Retrieve labware from the OT
Importing layout output maps from robot
There will be 1 final excel file with the name set in variable 'Final Map Name' from sheet 'GeneralVariables' with as many maps as final plates with the following structure of names: FinalMap followed by the PositionSlot where that final plate was
Layout maps will be in directory /data/user_storage of the robot where the protocol was run
For additional information on how to retrieve the maps from the OT-2 liquid handler:
Example
2h 31m
We want to perform a PCR mix preparation of 93 colonies and 2 controls that are in 2 source plates (40 colonies + 2 control samples in 1 plate and 54 colonies starting from the A4 well in the other plate but we dont want to take the sample in the well D6) with 2 primer sets. The first plate will have a map of identities(names) but the second one wont.
We are going to have a heater-shaker module available but not a thermocycler. We are going to set the shaker to a high RPMs so we want the maximum volume on the mixing tubes, the tube sthat will contain the PCR mix with primers, polymerase mix and water, to be 1mL
We will use a computer with a Windows 10 system
Prepare variable file
Excel temple filled and saved with the name VariablesPCR.xlsx
VariablesPCR.xlsx 10m
Upload custom labware to app
We are using a custom labware called vwrblueprintdepth105_96_wellplate_390ul and 3dprinted_opentrons_shaker_1.5mleppendorf that has been created with the labware creator that opentrons offer (https://labware.opentrons.com/create/)
3dprinted_opentrons_shaker_1.5mleppendorf.json 
vwrblueprintdepth105_96_wellplate_390ul.json We upload it to the opentrons app (make sure that is in the robot app) and the robot system as stated in the protocol in step Setting and Customizing OT-2 for LAP Entries
vwrblueprintdepth105_96_wellplate_390ul.zip 
3dprinted_opentrons_shaker_1.5mleppendorf.zip 2m
Export the variable file to the /data/user_storage folder in the robot
command line window with scp commands to transfer the variables .xlsx from our computer to the OT-2
For more information about sending files to the OT-2 Setting and Customizing OT-2 for LAP Entries
30s
Import the script
Import to the OT App the script that we have downloaded from the step (I named it Example-PCR.py)
Example-PCR.py Result of importing the Python script in the OT-App
As we can see, we have an error, but that is programmed because the script is meant to work in the robot but not in your computer
1m
Run the protocol in the robot that we have transferred the Excel file
Example-PCR.py -> Start setup -> Select robot in which we are going to run the protocol
If we do not have any errors, the output should look similar to the following pictures
Labware and liquid set-up layout
Volumes of the different reagents needed to perform the protocol
If we click to the different reagents and the labware where they are or go to Map view and click in the different labware with liquids we can see the distribution and volumes of each tube/well in the different labwares like the following pictures.
Position and volume of all the samples in the Plate in Slot 2 that are going to be picked to be mixed with the different sets of PCR mix
Positions and volume of the samples in the Plate in Slot 2 are going to be used as control
Position and volume of all the samples in the Plate in Slot 3 that are NOT going to be picked to be mixed with the different sets of PCR mix
Position and volume of the tube that is going to contain the polymerase mix
Position and volume of the tube that is going to contain MiliQ water
Position and volume of the tube that is going to contain 1 of the primers that are going to be in the final PCR sets
Positions of the final tubes containing the set of primers, in this case Primer 1 and Primer 2, in the heater-shaker situated in the Slot 1
30s
Turn the HEPA filter module
30s
Clean the platform of the robot that we are going to perform the protocol
2m
Prepare all reagents and labware in the places the App is showing and take into account the notes in step Notes
5m
Start run
Expected result
Final layout of source and final plates in the run
Content of A4 in the labware Final Plate 1 Slot 4 at the end of the run
Here, we will obtain the mix between the PCR mixes and the samples selected by the values in the variable file. These positions are seen in the images by the grey wells, and we can see the info of the plate and the media in the well A4 of the final plate on slot 4 in the second image.
2h
Retrieve labwares from the OT
5m
Retrieve the final maps that are going to be in the file. In this case, they will be called mapPCR-example.xlsx (name that is stated in the variable file in the variable' Final Map Name')
command line windows with the transfer command of the final file with the map(s) from the OT to our computer
mapPCR-example.xlsx For more information about how to transfer files from one system to another one, in this case from the OT to your computer, Setting and Customizing OT-2 for LAP Entries
1m
Protocol references
pBLAM1-x: standardized transposon tools for high-throughput screening (Synthetic Biology) https://doi.org/10.1093/synbio/ysad012
The Laboratory Automation Protocol (LAP) Format and Repository: A Platform for Enhancing Workflow Efficiency in Synthetic Biology (ACS Synth. Biol.) https://doi.org/10.1021/acssynbio.3c00397