OT-2 Custom Mixing with Single or Multi-Channel Pipette

Ana Mariya Anhel, Ángel Goñi-Moreno, Paula Mugica-Galan

Published: 2024-08-07 DOI: 10.17504/protocols.io.5jyl82686l2w/v1

Abstract

This protocol is created to help efficiently produce high-throughput plates with different reagents using either a single or multi-channel pipette controlled by an Opentrons OT-2 robot. By running this script, users can achieve a precise layout of reagents in specified volumes across the final plate(s) using a single-channel or multi-channel pipette based on a customizable Excel input file.

The protocol uses a Python script for the Opentrons OT-2 robot in conjunction with an Excel file that contains all the necessary parameters and will be processed by the Python script.

This Excel file contains customizable variables such as the number of final plates, transfer volumes, plate types, and the type of pipette used.

This method ensures the accurate distribution of reagents based on user-defined configurations, making the high-throughput process more efficient.

This protocol has been used in our laboratory to check how different quorum sensing systems work in P. putida KT2440. P. putida KT2440.

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

You can find the script and complementary information for this specific version of the protocol in this LAP entry link and GitHub Link to LAP entry documents

Steps

Files Preparation

Preparing Customized Template

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

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

GeneralVariables: These are variables mainly related to the labware to be used, optimization for the source plate, and sterility variables.
PipetteVariables: These are variables related to the pipettes to be used.
FinalPlatesVariables: variables related to the specifications of each final plate to be created, such as the maps associated with this final plate, the number of replicas to make and the type of pipette used to create each.
MapsReagents: These are the sheets that are established in variables in FinalPlatesVariables that define the layout of reagents to transfer/distribute to each one of the final cells
MapsVolumes: These are the sheets established in variables in FinalPlatesVariables that define the layout of volumes of each reagent defined in MapReagents to transfer/distribute to each of the final cells.

TemplateVariablesCustomMixing.xlsx

CustomMixingInstructions_v100.pdf

Note

The most updated Excel template can be found on the The most updated Excel template can be found on the LAPrepo Repository Page

1.1.

Fill the template with the corresponding values

1.2.

Save it with the name VariablesCustomMixing.xlsx

Note

The file should be spelt precisely as VariablesCustomMixing.xlsx or the Python script won't work correctly.

Setting the robot

Prepare the system of the robot to run the protocol

For this protocol to work, we need to transfer the file VariablesCustomMixing.xlsx to the directory /data/user_storage of the OT system we will use to perform the protocol.

Also, if we use custom labware, we must 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 must ensure the package openpyxl is installed in the robot system.

We can do this by following the protocol Setting and Customizing OT-2 for LAP Entries with the specifications in the text above.

Setting and Customizing OT-2 for LAP Entries

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 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 script version.

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

3.1.

Load the script in the App

Protocols -> Import -> Drag Python script

This protocol version was developed when the last version of LAP-CustomReagentMixingMultiSinglePip-OT2 was 1.0.0; the script is attached.

ScriptMixingReagents_v100.py

The name of the Python file is the user's choice; it will work with any name in the app.

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 the directory should be LAP-CustomReagentMixingMultiSinglePip-OT2, followed by the version.. The name of the directory should be LAP-CustomReagentMixingMultiSinglePip-OT2 , followed by the version.Also, the latest version of the script can be found at Also, the latest version of the script can be found at https://www.laprepo.com/repository/, with the same name as on GitHub., with the same name as on GitHub.

Software

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

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.

3.2.

Select Robot to Perform Script

Click in the protocol -> Start setup -> Choose the OT where the file VariablesCustomMixing.xlsx is -> Proceed To Set to overpour to account for any pipetting errors slightly up

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

If the protocol with the set variables cannot run, an error will occur during the simulation's run in the app after Start setup . Many errors are already contemplated and have a specific message that hints tothe user what could have gone wrong.

Note

The reagent volumes in the reservoirs for the multi-channel pipettes or tubes for the single-channel pipettes are precisely measured, so it is recommended to overpour slightly to account for any pipetting errors.

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 to prepare the experiment layout. That way, you reduce the probability of contamination (using the test plates and labware) and pipetting errors (position check).

Run Protocol in OT

4.1.

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

4.2.

Labware position check is performed (if required)

4.3.

Turn the HEPA Filter on

4.4.

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.

4.5.

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

4.6.

Start Run

The procedure that the robot is going to do is mainly divided into 2 parts:

Distribute reagents with single-channel if any final plate is set to be created with it.
Distribute reagents with multi-channel if any final plate is set to be created with it.

Citation

One or more plates, with the reactive-volume layout set by the user in the Excel input file.

After-running

Retrieve labware from the OT

Example

We want to create three plates: 1 with a single-channel pipette and 2 with a multi-channel pipette.

The two plates filled with the multi-channel will have different concentrations of IPTG and certain bacteria. At the same time, the single-channel pipette will create a plate with varying combinations of reagents.

The multi-channel reservoir source's optimization will be low, and the reservoir will have 15 columns and 1 row.

The tip will be changed every time a reagent is changed, and the liquids will be dispensed at the top so the source reagents are not contaminated.

We will use a computer with a Windows 10 system.

6.1.

Prepare variable file

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

VariablesCustomMixing.xlsx

VariablesCustomMixing.xlsx

6.10.

Start Run

Citation

After the run has finished, we should obtain 3 final plates with the reagents and volumes described in the input Excel file.

Final content of C3 in PlateMulti1 located in Slot 1

Final content of A6 in PlateMulti2 located in Slot 2

Final content of A7 in PlateSingle1 located in Slot 3

6.11.

Retrieve Labware from the robot

6.2.

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

We upload it to the opentrons app (make sure that it is in the robot app) and the robot system as stated in the protocol in step

6.3.

Export the Excel file to the robot

Export the VariablesCustomMixing.xlsx file to the /data/user_storage directory in the robot where the script is going to be played following the instructions in

6.4.

Import the script to the robot

ScriptMixingReagents_v100_example1.py

Result of importing the Python script in the OT-App

Note

An error during the simulation of the script in the app is normal. The script is meant to work in the opentrons robot, not on your computer.

6.5.

Run the protocol

ScriptMixingReagents_v100_example1.py ScriptMixingReagents_v100_example1.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