OT-2 Protocol to transfer volume from several plates to a single plate

Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno

Published: 2023-12-23 DOI: 10.17504/protocols.io.6qpvr4o62gmk/v2

Abstract

This protocol is meant to transfer samples from different plates to a single or fewer final plates than the source number, in other words, merge samples of different source plates in a final plate.

The output of running this script will be the final plate(s) with samples and a reactive (optional), and the corresponding map(s) with the original identities of the samples which will be given by the user in the input file.

This protocol uses a python script for an Opentrons 2 robot and an excel file containing the required variables to set the number of samples, volumes of transfer, type of plates, 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

This protocol is a set of instructions or description of the LAP repository entry LAP-NplateMerging-OT2-1.0.0

Before start

It is important to annotate the location of the source plate in the OT-2 slots, the identity of the samples is provided by excel sheets in the input variable and if 2 samples, within a plate or between different source plates has the same name, they will be indistinguishable in the final plate (s)

Steps

Files Preparation

Preparing Customized Template

Preparing the template (a .xlsx) with the specific variables for each experiment.

Attached there is a template of the variable file with several sheets and a PDF file explaining each variable:

GeneralVariables: variables related to the labware to be used
PipetteVariables: variables related to the pipettes to be used
PerPlateVariables: variables related to the specifications of each source plate
Maps (Optional): sheet(s) with the names of the samples in the source plates. These will be reflected in the final plate map --> not included in the template but needs to be included and have the same names as established in the variable Name Sheet Map Identifiers from the PerPlateVariablesSheet Name Sheet Map Identifiers from the PerPlateVariablesSheet

Template-VariablesMergeSamples.xlsx

MergeSamplesInstructions.pdf

1.1.

Fill the template with the corresponding values

1.2.

Save it with the name VariablesMergeSamples.xlsx

Note

The file should be spelt exactly VariablesMergeSamples.xlsx or the Python script won't work correctly

Transferring VariablesMergeSamples.xlsx file to Robot VariablesMergeSamples.xlsx file to Robot

Transfer the VariablesMergeSamples.xlsx to the directory /data/user_storage of the OT robot

that we are going to 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 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 VariablesMergeSamples.xlsx to the OT system.

We need to perform the following command

#Passing Files to OT 
scp -i [ot_key] [file] root@[IP_OT]:/data/user_storage

Note

You could face difficulties transferring files in MacOS Ventura (13) and Sonoma (14). These problems can be solved by adding the argument -O (uppercase o) to the command #Transferring files to OT (MacOS 13 and 14)scp -Oi [ot_keypath] [file path] root@[OP_robot]:/data/user_storage

Windows

There are several ways to send files from a Windows to a Linux (for example, with a virtual machine or Windows Powershell in the latest versions of Windows).

Here, we will use FileZilla (https://filezilla-project.org/download.php?type=client).

Go to File -> Site Manager -> New Site -> Change Protocol to SFTP . Then, introduce in Host the OT IP, change the Logon Type to key file, change User to root and give the directory where the ot key is. It should look something like this

Example of setting the FileZilla to transfer files from Windows (our computer) to Linux (OT)

Then press Connect , and we will have a connection between our computer and the robot.

After this connection, we should be able to move the file VariablesMergeSamples.xlsx (in our computer) to the directory /data/user_storage in the robot.

This method can be used as well in some Linux and MacOS

Note

Take into account that the IP of the robot could change, so it is possible that it will be needed 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 that you are using is not OT official or is not included in the OT app

3.1.

Creation of .json file

The description file can be obtained by describing the labware dimensions at https://labware.opentrons.com/create/

3.2.

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

Citation

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

3.3.

Transfer labware files to the robot

If you are using the entry LAP-NplateMerging-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.

#Transferring the custom labware to OT (Linux)
scp -i [ot_key] -r [directory_custom_labware] root@[IP_OT]:/data/labware/v2/custom_definitions/custom_beta

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 RobotOS

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 protocolIf the package is not installed, an error will appear when running the script in the robot. While simulating the script in the app, this error will appear, but you can ignore it

4.1.

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

#Connect to Linux based OT via ssh 
ssh -i [path ot_key] root@[Robot_IP]
```In Windows you can do this command in Windows Powershell



<Note title="Citation" type="success" ><span>If the connection has been successful you should obtain a screen similar to the following image</span><span></span><img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.6qpvr4o62gmk/pd4vbxsu720.jpg" alt="Drawing obtained when entering an OT-2 system" loading="lazy" title="Drawing obtained when entering an OT-2 system"/></Note>

4.2.

Install the package

Once inside the robot's system, you need to run the following command

#Install openpyxl package (Linux 4.14.74-v7)
pip install openpyxl

Note

For more information about installing packages in the opentrons robots, check the following Opentrons page: 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

Running 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 until version 7.0.2Indications may vary from version to version of the opentrons App and the version of the script.

Software

Value	Label
Opentrons App	NAME
Windows >=10, Mac >=10	OS_NAME
, Ubuntu >=12.04	OS_VERSION
https://opentrons.com/ot-app/	REPOSITORY
Opentrons	DEVELOPER

5.1.

Load the script in the App

Protocols -> Import -> Drag Python script

Note

The last script version can be found at 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). The name of the directory should be LAP-NplateMerging-OT2 followed by the version. (the name of this file is the user's choice). The name of the directory should be LAP-NplateMerging-OT2 followed by the version.As well we can find the latest version of the script at 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 with the same name as in GitHub

Software

Value	Label
LAP Repository	NAME
www.laprepo.com	REPOSITORY
https://biocomputationlab.com/	DEVELOPER

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

5.2.

Select Robot to Perform Script

Click in the protocol -> Start setup -> Choose the OT where the file VariablesMergeSamples.xlsx is -> Proceed To Setup

After clicking on Proceed to Setup, you should obtain, the positions of the labware in the Labware tab and in the Liquids tab, the reagents and their corresponding volume.

Citation

A liquid setup should look like the following image, where you can find the samples in the initial plates and the reactives in the Falcon labware

Liquid Set-up Example of a Samples Merge Protocol

A labware setup should look like the following image, where you can find the initial and final plates, the falcon labware (if needed) to store the reagent and the corresponding tip rack(s)

Labware Set-up example of a Samples Merge Protocol

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 the final plate(s). The volume of the reagents is exactly what is needed, so it is suggested to pour always more to take in 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

6.1.

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

6.2.

Labware position check is performed (if required)

6.3.

Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces

Note

Check the Opentrons page 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 for more information about cleaning the OT-2 robot with the proper materials

6.4.

Set the labware and reagents as shown in the OT-App

6.5.

Start Run

The procedure that the robot is going to do is mainly divided into 3 parts:

Select which samples, taking into account the variable Type of Sample Selection
(Optional) Distribute reactive(s)
Distribute selected samples

Citation

One or more plates , with a reactive mixed with the samples from different sources or only the samples. In addition to an Excel file where every sheet is the map of a final plate located in the folder /data/user_storage /data/user_storage that will give the position in these plates with their identifiers (the name given in the maps of each source plate in the input variable file)

After-Running

Retrieve labware from the OT

Import map from robot

There will be a map with the name set in the variable Name File Final Map set in the sheet GeneralVariables followed by the extension .xlsx: [ Name File Final Map ] .xlsx

This file will be located in the directory /data/user_storage in the robot where the script has been run.

To retrieve the file, we can and reproduce it by transferring the files from the robot to the computer.

#Transferring files from OT to computer (Linux, macOS)
scp -i [path_ot_key] root@[IP_robot]:/data/user_storage/[name_map].xlsx [ultimate_path_computer]

Citation

The sheets in the final map contain the identity of the samples selected in the places where they have been placed/distributedThese identities are going to be the names given in the map Excel sheets from the input file

Example

We want to merge 16 samples from 5 source plates in a different way, 1 of them selecting samples from back to front, 2 in a random way and 2 from front to back.

Our final plate will have half the plate already occupied by other samples , and we want to add to all the samples water , so we need to take into account those details

We will use a computer with a Windows 10 system

9.1.

Prepare variable file

Excel template that we can find filled and saved with the name

VariablesMergeSamples.xlsx

VariablesMergeSamples.xlsx

9.10.

Start run

Citation

Final layout of source and final plates in the run

Example of the content of A8 in the labware Final Plate 1 Slot 6

Here we will obtain the mix between the volume of the reactive, in this case, water, 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 reactive in the plate on slot 6 in the second picture

9.11.

Retrieve labwares from the OT

9.12.

Retrieve the final maps, in this case, the map file will be called example-mapMerge.xlsx (name that is stated in the variable file in the variable Name File Final Map )

Command line windows with the transfer command of the final plate(s) map(s) file from the OT to our computer

example-mapMerge.xlsx

9.2.

Upload custom labware to app

We are using a custom labware called vwrblueprintdepth105_96_wellplate_390ul that has been created with the labware creator that opentrons offers (https://labware.opentrons.com/create/)

vwrblueprintdepth105_96_wellplate_390ul.json

Upload it to the labware and make sure it is loaded in the app

List of custom labware recorded in the Opentrons App

9.3.

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

9.4.

Because we are using version 1.0.0 of the script in this example, we will transfer the directory of the labware as well (here we have attached a zip, but it is the folder that must be transferred, not the zip)

vwrblueprintdepth105_96_wellplate_390ul.zip

#Transferring the used custom labware to OT (Linux)
scp -i [ot_key] -r vwrblueprintdepth105_96_wellplate_390ul root@[IP_OT]:/data/labware/v2/custom_definitions/custom_beta

9.5.

Import the script that we have downloaded from the step (I named it Example-ScriptMergePlates . py ) to the OT-App

Example-ScriptMergePlates.py

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

9.6.

Run the protocol in the robot that we have transferred the Excel file

Example-ScriptMergePlates.py Example-ScriptMergePlates.py -> Start setup -> Select the 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

Volumes of the needed liquids to perform the protocol

9.7.

Turn the HEPA filter module

9.8.

Clean platform of the robot that we are going to perform the protocol

9.9.

Prepare all reagents and labware in the places the App is showing and take into account the notes in step

OT-2 Protocol to transfer volume from several plates to a single plate

Abstract

Before start

Steps

Files Preparation

Prepare RobotOS

Running Protocol

After-Running

Example

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