Dec 22, 2023
Version 2
OT-2 PCR sample preparation protocol V.2
- Ana Mariya Anhel1,
- Lorea Alejaldre1,
- Manuel Gimenez1,
- Ángel oñi-Moreno1
- 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
- Ángel oñi-Moreno: [email protected]
Protocol Citation: Ana Mariya Anhel, Lorea Alejaldre, Manuel Gimenez, Ángel oñi-Moreno 2023. OT-2 PCR sample preparation protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpyznl5b/v2Version 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: November 13, 2023
Last Modified: December 22, 2023
Protocol Integer ID: 90844
Keywords: PCR sample preparation, automation, opentrons, pcr sample preparation protocol, pcr sample, throughput workflow for the genotypic characterization, samples preparation of pcr plate, transposon library variant, pcr, python script for an opentron, bacterial genome, pcr plate, different primer set, description of the lap repository entry lap, protocol modular to reactive, more primer set, combination of primer, number of primer, polymerase mix, samples preparation, protocol, lap repository entry lap, primer, set of instruction, laboratory, correct introduction of the transposon, source plates the master mix, python
Funders Acknowledgements:
Comunidad de Madrid
Grant ID: Y2020/TCS-6555, 2019-T1/BIO-14053
MCIN/AEI
Grant ID: CEX2020-000999-S, PID2020-117205GA-I00
European Research Council
Grant ID: 101044360
Abstract
This protocol is meant to perform samples preparation of PCR plates for 1 or more primer sets (defined number of primers per set established by the user) to the same samples, i.e, we will have for all the selected samples (we can set the initial well and number of samples) of all source plates the master mix with different primer sets (combination of primers, water and polymerase mix) in the final plates.
To run this protocol is needed a python script for an Opentrons 2 robot and an excel (.xlsx) file with several variables making the protocol modular to reactives, volumes of transfer, type of plates, number of primers per set, etc.
In our laboratory, this protocol has been used as part of the "High-throughput workflow for the genotypic characterization of transposon library variants" also available in protocols.io to prepare PCR samples for sending to sequencing and for checking the correct introduction of the transposon in the bacterial genome.
Guidelines
This protocol was developed with python 3.7.1, OT App Software Version 6.3.1 and API level version 2.14 in a Linux 4.14.74 system (these are the OT-2 specifications).
In the script several packages are used: pandas (0.25.3), openpyxl (3.1.2), math, random and numpy (1.15.1)
It 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 library variants.
The maximum number of primer sets that can be incubated with a full 96-well plate having a heater-shaker module set as True, the thermocycler set as False, the replace tip rack variable set as False is 3. In this scenario, 3 final plates will be created
Materials
Software
- Python 3.7.1
- opentrons software version 6.3.1
- python packages: pandas (0.25.3), openpyxl (3.1.2), numpy(1.15.1), math, random
- OT App
- Excel
OT-2 Labware
- Opentrons Tip racks
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
Troubleshooting
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.
Template-VariablesPCR.xlsx21KB 
PCRInstructions.pdf211KB Fill the template with the corresponding values
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 file to Robot
Transfer the VariablesPCR.xlsx to the directory /data/user_storage of the OT robot that we will use to perform the protocol.
Note
Before transferring any file to the OT, we need to know the IP of the robot.
This can be obtained in the Networking section of the Device that we will use.
To obtain this info go to OT-App -> Devices -> Chosen Robot (three dots) --> Robot Settings -> Networking
In this tab, you can see 2 types of IP; one is shown if both the robot and you are connected to the same Wifi, and the other is shown if the computer and the robot are connected via USB. Both connections can be used for this step.
Note
To connect to the robot, an OT-key should have been previously generated, and it is done with the ssh-keygen command and transferring the public key to the OT.
For more information about how to generate and set the connection between your computer and the Opentrons robot, visit https://support.opentrons.com/s/article/Setting-up-SSH-access-to-your-OT-2
Here, we present a summary of how to transfer the files in 3 Operative Systems: Windows, MacOS and Linux.
MacOS/Linux
We will use the command line with scp to transfer the file VariablesPCR.xlsx to the OT system.
We need to perform the following command
Command
File passing from linux (our computer) to linux (OT raspberry)
Passing Files to OT
Note
You could face difficulties transferring files in the MacOS Ventura (13) and Sonoma (14). These problems were solved by adding the argument -O (uppercase o) to the command
Command
Transferring files to OT (MacOS 13 and 14)
Windows
There are several ways to pass files from a Windows to a Linux (for example, with a virtual machine or Windows Powershell in the latest versions of Windows).
Go to File -> Site Manager -> New Site -> Change Protocol to SFTP. Then, introduce in Host the OT IP, change the Logon Type to Windowskey file, change the user name to root and give the directory where the OT key is. It should look something like this
Then press connect, and we will have a connection between our computer and the robot.
After this connection, we should be able to move out VariablesPCR.xlsx (in our computer) to the directory /data/user_storage in the robot.
This method of transferring files can also be done in Linux and MacOS.
Note
Take into account that the IP of the robot could change, so it may be necessary to change the host in these connections from time to time.
Adding the custom labware
There is only a need to do this step when the labware you are using is not OT official or included in the OT app.
Creation of .json file
The description file can be obtained by describing the labware dimensions at https://labware.opentrons.com/create/
Uploading files to the OT App
In the OT app, we need to perform the following route: Labware -> Import -> Choose File -> Select file we have created in step 3.1
Expected result
After uploading the labware you should be able to see the new labware in the Labware tab of the OT App, all custom labware can be found more easily in the category Custom Labware
Transfer labware files to the robot
If you are using the entry LAP-PCR-OT2-1.0.0 and custom labware,. an additional step is needed, which is transferring a folder with the custom labware
We need to create for our custom labware a folder with the API name containing the description file (.json) called 1.json and then transfer that folder to the robot's folder /data/labware/v2/custom_definitions/custom_beta in a similar way as in the Step 2 but with the difference that is a directory that needs to be transferred and not a file.
Command
Transferring the custom labware to OT (Linux)
Note
We do not need to execute this part every time the protocol is used, only when that labware is not included in the OT official labware and these directories are not in the robot
Prepare Robot OS
Install needed packages
This script needs the package openpyxl, which is not installed by default in the OT-2 robots
Note
This step is only needed if the package is not installed in the robot, not every run of the protocol
If the package is not installed, an error when running the script in the robot will appear, while simulating the script in the app, this error will appear but you can ignore it
Connect to the robot
to find the IP of the robot in which you want to run the script
To connect to the robot, you can do it via ssh with the following command
Command
Connect to Linux based OT via ssh
In Wsuccessfulindows, you can do this command in Windows Powershell
Expected result
If the connection has been successful you should obtain a screen similar to the following image
Drawing obtained when entering an OT-2 system
Install the package
Once inside the robot's system,, you need to run the following command
Command
Install openpyxl package (Linux 4.14.74-v7)
Note
For more information about installing packages in the opentrons robots, check the following Opentrons page: https://support.opentrons.com/s/article/Using-Python-packages-in-Python-API-protocols
Run protocol
Load script in OT-App
Now that we have transferred the variable files to the robot, we can import the script and run it in the selected robot
Note
This whole step has been developed with version 6.3.1 of the OT-App and has been tested in versions up to 7.0.2
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
Load the script in the OT app
Protocols -> Import -> Drag Python script
Note
The last script version can be found at https://github.com/BiocomputationLab/LAPrepository/tree/main/LAPEntries (the name of this file is the user's choice) in the directories with the name LAP-PCR-OT2 followed by the version.
As well we can find the latest version of the script at https://www.laprepo.cbgp.upm.es/repository/ with the same name as in GitHub
Note
The App with version 6.3.1 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 VariablesPCR.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.
Expected result
A labware setup should look like the following image, where you can find the initial and final plates, the Eppendorf labware to store the reagents, the corresponding tips and, if included, the location of the heater-shaker (s) and thermocycler. The latter will always have the exact location in the OT-2
Labware set-up of an example of a PCR run with both variables 'Presence Thermocycler' and 'Presence Heater-Shaker' set as True
Labware set-up of an example of a PCR run with both variables 'Presence Thermocycler' and 'Presence Heater-Shaker' set as False
Expected result
A liquid setup should look like the following images, where you can find the samples in the initial plates and the different reagents in the Eppendorf labware with their respective volume
Liquid set up of a reagents tube rack
Note
The volume of the initial samples is established to be 90% of the max volume of the well, but it 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 exactly what is needed, so it is suggested to pour always more to take into account the error of pipetting
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 the test plates and labware) and pipetting errors (position check).
Run Protocol in OT
Make sure the needed calibrations are done
Pipettes, tip racks and tip length calibrations need to be done for the items used in this run
Labware position check is performed (if needed)
Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces
Note
Check the Opentrons page https://support.opentrons.com/s/article/Cleaning-your-OT-2? for more information about cleaning the OT-2 robot with the proper materials.
Set the labware and reagents as shown in the OT-App
Start Run
The procedure that the robot is going to do is mainly divided into 6 parts:
- Creation of mix(es) transferring primers, water and polymerase to new tube(s)
- Mixing with either a pipette or heater-shaker
- Distribute mix to all plates
- Distribute samples to the corresponding wells (as many transferring of 1 sample as the number of primer sets)
- Generate identity maps to be exported
- (Optional) PCR program for thermocycler module
Expected result
One or more plates where there is a mix between the sample (DNA-template) and the set of primers, each combination of DNA template set of primer in one well
The distribution of each primer will be sequential. In other words, all the DNA templates with the first set of primers will be in the first N-wells, then all the DNA templates with the second set of primers will be in the following N-wells and so on.
A sheet for every final plate will be created as well in an Excel file with the given name in the shee GeneralVariables in the variable "Final Map Name" followed by the extension .xlsx in the folder /data/user_storage of the robot where we run the script.
After-running
Retrieve labware from the OT
Importing map from robot
To retrieve the file we can and reproduce it by transferring the files to the computer.
They will be in the directory /data/user_storage. It will be a file with an extension .xlsx and have the name provided in the input variable file
Command
Transferring files from OT to computer (Linux, macOS)
Expected result
The map(s) contains a table for each final plate in the run
Each table will have the identity or name of the sample and the set of primers that are mixed in that well. If a map has been provided for the source plate, it will have that name as an identity. If not, the identity will be the well in the slot that the source plate was in the run of the script.
Example
2h 31m
We want to perform a PCR mix preparation of 94 colonies and 2 controls that are in 2 source plates(40 first colonies + 2 control samples in 1 plate and 54 colonies from the A4 well in the other plate) with 2 primer sets.
We are going to have a heater-shaker module available but not a thermocycler.
1 of the source plates will have a map of identities, but the other will not have it.
We will use a computer with a Windows 10 system
Prepare variable file
Excel temple filled and saved with the name VariablesPCR.xlsx
VariablesPCR.xlsx23KB 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/)
vwrblueprintdepth105_96_wellplate_390ul.json11KB 3dprinted_opentrons_shaker_1.5mleppendorf.json3KB Upload it to the labware and make sure it is loaded in the app
List of custom labware recorded in the Opentrons App
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
2m
Because we are using version 1.0.0 of the script in this example, we will transfer the directory of the labwares as well (here we have attached a zip, but it is the folder the one that must be transferred, not the zip)
vwrblueprintdepth105_96_wellplate_390ul.zip1KB 3dprinted_opentrons_shaker_1.5mleppendorf.zip1KB Command
Transferring the used custom labware to OT (Linux)
Command
Transferring the used custom labware to OT (Linux)
2m
Import the script that we have downloaded from the step (I named it Example-ScriptPCR.py) to the OT-App
Example-ScriptPCR.py81KB The result of importing the Python script into 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
30s
Run the protocol in the robot that we have transferred the Excel file
Example-ScriptPCR.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
1m
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
Example of the content of C5 in the labware Final Plate 1 Slot 4
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 image by the grey wells, and we can see the info of the plate and the media in the plate on slot 4 in this case
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 example-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.xlsx10KB 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