OT-2 Media dispensing and culture inoculation protocol

Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno

Published: 2023-12-23 DOI: 10.17504/protocols.io.q26g7yb3kgwz/v2

Abstract

This protocol is meant to distribute reactive(s) into well plates with a single channel pipette, and then transfer culture samples from source plate(s) to these plates with a multi-channel pipette.

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 to prepare cultures plates to perform a subsequent counter-selection

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

Before start

Note that the source and final 96-well plates will follow the same order (If the wells of a column of the source plates has empty wells the multichannel will aspirate in that well) but media will not be dispensed in those corrresponding final wells

Steps

0.36.

Select Robot to Perform Script

Click in the protocol > Run Protocol > Choose the OT where the file VariablesPlateIncubation.xlsx is > Proceed To Setup

After clicking in Proceed to Setup you should obtain the positions of the labware, in the Labware Setup tab, and the reactives, with their correspondant volume, in the Liquid Setup tab.

In case that the protocol with the set variables is not possible to run, an error will occur during the run. A lot of errors are contemplated already and have a specific message that give a hint of what could have gone wrong.

Citation

A labware setup should look like the following image where you can find the initial and final plates, the falcon labware to store the reagents and the corresponding tips

Labware Set-up example of Incubation Protocol

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

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 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).

Files Preparation

Preparing Customized Template

Preparing the template (a .xlsx) with the specific variables for each experiment and a .pdf that contains the instructions on how to fill the template

Here we attach one Excel with several sheets:

GeneralVariables: variables related mainly to the labware that is going to be used
PipetteVariables: variables related to the pipettes that are going to be used
PerPlateVariables: variables associated with the specifications of each source plate

Template-VariablesPlateIncubation.xlsx

InoculationInstructions.pdf

1.1.

Fill the template with the corresponding values

1.2.

Store it as VariablesPlateIncubation.xlsx

Note

The file should be spelt precisely as VariablesPlateIncubation.xlsx or the Python script won't read it

Transferring file to Robot

Transfer the VariablesPlateIncubation.xlsx to the directory /data/user_storage of the OT system 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 are going toOT-App -> Devices -> Chosen Robot (three dots) --> Robot Settigns --> 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

An OT-key should have been previously generated to connect to the robot, 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 OS: Windows, MacOS and Linux .

MacOS/Linux

We will use the command line with scp to transfer the file VariablesPlateIncubation.xlsx to the OT system.

We need to perform the following command

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

Note

You could face difficulties transferring files in the 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 Logon Type to key file, change User to root and give the directory where the OT-key file is. The final window should look something like the following picture

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 transfer VariablesPlateIncubation.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 is possible that the host in these connections will need to be changed 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.

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 quickly in the category Custom Labware .

3.3.

Transfer labware files to the robot

If you are using the entry LAP-CellMediaInoculation-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 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.If the package is not installed, an error will appear when running the script in the robot. This error will appear while simulating the script in the app, 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.q26g7yb3kgwz/pd7ubxsu718.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

Run 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 with version 6.3.1 of the OT-App and has been tested in versions up to 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) in the directories with the name LAP-CellMediaInoculation-OT2 followed by the version. (the name of this file is the user's choice) in the directories with the name LAP-CellMediaInoculation-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 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 at what could have gone wrong.

Citation

A labware setup should look like the following image, where you can find the initial and final plates, the Falcon labware to store the reagents and the corresponding tips

Labware Set-up example of the Incubation Protocol

A liquid setup should look like the following image, where you can find the samples in the initial plates and the reactions in the 15-mL tube labware

Liquid Set-up Example of the Incubation Protocol

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 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 needed)

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 2 parts:

Distribute each reactive to the final plate(s) with the single-channel pipette
Distribute samples to the final plate(s) with the multi-channel pipette

Citation

One or more plates with different reagents and same samples as set in the input variable fileThis set of plates will be given for each source plate the user has provided with the reagents the user has provided ,as well

After-Running

Retrieve labware from the OT

Example

We want to transfer samples from 2 source plates, with 96 and 50 samples , to different antibiotic plates, one per antibiotic and source plate.

The first one will be mixed with Ampicillin and Kanamycin. The second plate will be mixed with Ampicillin and Gentamycin .

We will use a computer with a Windows 10 system.

8.1.

Excel template that we can find filled with the name VariablesPlateIncubation.xlsx

VariablesPlateIncubation.xlsx

8.10.

Start Run

Citation

Final labware layout in which the grey wells are a mix of the correspondent reactive with the sample

Example of the content of A1 in the labware Sample Plate 'Plate 1' with Km Slot 4

Here, we will obtain the mix between the volume of media and the samples set in the variable file in the final plates. These positions are seen in the first image by the grey wells, and we can see the info on the plate and the media in the name of the labware and an example of the composition of 1 well in the second picture.

8.11.

Retrieve labwares from the OT

8.2.

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 ensure it is loaded in the app.

List of custom labware recorded in the Opentrons App

8.3.

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

8.4.

Export the variable file to the /data/user_storage folder in the robot.