Jul 24, 2024
Version 3
OT-2 Protocol to transfer volume from several plates to a single plate V.3
- 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 Goñi-Moreno: angel.goni@cnb.csic.es;
Protocol Citation: Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno 2024. OT-2 Protocol to transfer volume from several plates to a single plate . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4o62gmk/v3Version created by Ana Mariya Anhel
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 20, 2023
Last Modified: July 24, 2024
Protocol Integer ID: 101406
Keywords: OT-2, Opentrons, Media transfer, 96-well
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 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-OT2-2.0.0
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
The major changes from previous version are:
- Change of name variable API Name Rack 15mL Falcon Reactives which is called now API Name Rack Falcon Reactives
- The program now accepts falcon tubes and tube racks of 15ml or 50ml. It does not accept mix tube racks.
- Description of robot and protocol setup in a separate protocols.io entry (Setting and Customizing OT-2 for LAP Entries)
- The page(s) that corresponds to the map(s) set in the variable 'Name Sheet Map Identifiers' of the page PerPlateVariables need to have the column and row names corresponding to the ones of the labware set in the variable 'API Name Source Plate' of the sheet GeneralVariables
Guidelines
This protocol was developed with python 3.7.1, OT App Software Version 7.0.2 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 as part of the High-throughput workflow for the genotypic characterization of transposon library variants.
The maximum number of 96-well plates per run is 8 source plates to 1 final plates dispensing some media (need of 1 falcon tube rack), 1 pipette is required for all the volumes and the replace of tip racks is allowed (set as True in the input variable file)
Materials
Software
- Python 3.7.1
- opentrons software version 7.0.2
- 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
- Opentrons Falcon tube rack
Equipment
Opentrons 15 Tube Rack with Falcon 15 mL Conical
NAME
OT Tube Rack
TYPE
Opentrons
BRAND
-
SKU
LINK
- 15mL Falcon tubes
Equipment
Falcon® Conical Centrifuge Tubes 15mL
NAME
Flaocn Tube
TYPE
Falcon
BRAND
352096
SKU
LINK
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
It is important to note 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. It should be noted that If 2 samples within a plate have the same name, they will be indistinguishable in the final plate(s)
Files Preparation
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
MergeSamplesInstructionsv200.pdf 
TemplateMergeSamples.xlsx Note
Fill the template with the corresponding values
Save it with the name VariablesMergeSamples.xlsx
Note
The file should be spelt exactly VariablesMergeSamples.xlsx or the Python script won't work correctly
Setting the robot
Setting the robot
Prepare the system of the robot to run the protocol
For this protocol to work we need to transfer the VariablesMergeSamples.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
Protocol
NAME
Setting and Customizing OT-2 for LAP EntriesCREATED BY
biocomp.cbgp Biocomputation Lab
Running Protocol
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 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
SOURCE LINK
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-NplateMerging-OT2 was the 2.0.0 which script you can find attached
ScriptMergePlates_v200.py The name of the python file is user's choice, it will work with any name in the app.
Note
The last script version can be found at https://github.com/BiocomputationLab/LAPrepository/tree/main/LAPEntries. 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 https://www.laprepo.com/repository/ with the same name as in GitHub
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 VariablesMergeSamples.xlsx is -> Proceed To Setup
After clicking on Proceed to Setup, you should obtain, the positions of the labware in the Labware and in the Liquids tabs, the reagents and their corresponding volume.
In case the protocol with the set variables cannot run, an error will occur during the run of that simulation. 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 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
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 required)
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 3 parts:
- Select which samples, taking into account the variable Type of Sample Selection
- (Optional) Distribute reactive(s)
- Distribute selected samples
Expected result
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 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
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 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 Linked protocol and reproduce it by transferring the files from the robot to the computer.
Take in account that files overwrite, so if you have given the same name to the final map in 2 consecutive runs, you will get only the results of the last run.
Expected result
The sheets in the final map contain the identity of the samples selected in the places where they have been placed/distributed
These identities are going to be the names given in the map Excel sheets from the input file
Example
Example
1h 5m
1h 5m
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
Prepare variable file
Excel template that we can find go to step #1 filled and saved with the name
VariablesMergeSamples.xlsx
VariablesMergeSamples.xlsx 10m
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.json11KB 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 (this is a zip file because needed to be compressed to upload to protocols.io but what needs to be transferred to the robot is the folder inside of the zip file)
2m
Export the variable file to the /data/user_storage folder in the robot
For more information about sending files to the OT-2 Setting and Customizing OT-2 for LAP Entries
command line window with scp commands to transfer the variables .xlsx from our computer to the OT-2
2m
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
30s
Run the protocol in the robot that we have transferred the Excel file
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
Labware and liquid set-up layout
Volumes of the needed liquids to perform the protocol
1m
Turn the HEPA filter module
30s
Clean 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 go to step #3.2 Notes
5m
Start run
Expected result
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
35m
Retrieve labwares from the OT
5m
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.xlsx9KB For more information about how to retrieve files from the robot Linked Protocol
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