Sep 11, 2024
Version 3
OT-2 Media dispensing and culture inoculation protocol 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 Media dispensing and culture inoculation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7yb3kgwz/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: October 03, 2023
Last Modified: September 11, 2024
Protocol Integer ID: 100118
Keywords: automation, opentrons, OT-2, media dispensing, culture inoculation
Funders Acknowledgement:
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 distribute reactive(s) (i.e bacterial culture media) into final 96-well plates with a single channel pipette and then transfer culture samples from source plate(s) to these plates with a multi-channel pipette.
In our laboratory, this protocol has been used as part of the "High-throughput workflow for the genotypic characterization of transposon insertion library variants" also available in protocols.io to prepare cultures plates to perform a subsequent counter-selection.
To run this protocol a python script in LAP format for an Opentrons OT-2 robot and an excel file with variables such as culture volume, transfer volume etc... This protocol provides a set of instructions or description of the LAP repository entry LAP-CellMediaInoculation-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:
- New variables are added to GeneralVariables sheet: Change Tip In Media Distribution, Change Tip In Sample Transfer, Position Transfer Sample, Touch Tip After Transferring Sample, Touch Tip In Distribution Media, Mixing Volume Before Sample Transfer (uL), Number Times of Mixing Volume and Flow Rate Mixing.
- Change of name variable Name 15mL Tuberack which is called now Name Tuberack
- New variables are added to PerPlateVariables sheet: Number of Replicas, Only Media(s) Plate Creation and Only Sample(s) Plate Creation
- The program now accepts falcon tubes of 15ml and 50ml
- Description of robot and protocol setup in a separate protocols.io entry (Setting and Customizing OT-2 for LAP Entries)
For all the new variables you can find more information in the PDF attached in the first step of this protocol or in the LAP entry of this program in the LAP entry link given before
Guidelines
This protocol was run in a python 3.7.1, OT App Software Version 7.0.2 and opentrons 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
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. LB and M9-citrate media with either kanamycin, gentamicin, streptomycin or ampicillin haven been successfully run.
The maximum number of 96-well final plates per run given 1 source plate, 2 different types of tip racks (with the replacement of the tip rack set as True) is 7 final plates (use 1 falcon tube rack and 2 tip racks)
Materials
Software
- Python 3.7.1
- opentrons software version 7.0.2
- python packages: pandas (0.25.3), openpyxl (3.1.2), 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) 1000uL
NAME
Opentrons Single Channel Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
8 Channel Electronic Pipette (GEN2) 20uL
NAME
Multi channel pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Safety warnings
It is important to use HEPA module to work in sterility
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
Files Preparation
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
CellAndMediaInoculationInstructionsv200.pdf 
TemplateVariablesCellInoculation.xlsx Note
Fill the template with the corresponding values
Store it as VariablesPlateIncubation.xlsx
Note
The file should be spelt precisely asVariablesPlateIncubation.xlsx or the Python script won't read it
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 VariablesPlateIncubation.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
Run Protocol
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 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-CellMediaInoculation-OT2 was the 2.0.0 which script you can find attached
ScriptPlateGenerationAndIncubation_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 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 LAPrepo Repository Page 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 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 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 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 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 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
Expected result
One or more plates with different reagents and same samples as set in the input variable file
This set of plates will be given for each source plate the user has provided with the reagents the user has provided, as well, the number of them is dependent of how many replicas the user has set
After-Running
After-Running
Retrieve labware from the OT
Example
Example
1h
1h
We have 2 source plates, only with 96 samples and the other with 50.
With the first source plate we want to create 1 plate with only samples from the source plate. With the second source we want to create 2 final plates, 1 with Ampicilin and another with Kanamycin inoculated with these samples starting to transfer samples from the 3rd column of the source plate. As well, we want to create 1 replica of those 2 plates.
Finally, we want to create 1 additional plate without only Ampicilin in 73 wells starting from the well C2
We need to change the tip during the sample transferring to be every time the pipette aspirates from the source plate but we just need the tip during the distribution of the media to change everytime it changes of reagent.
We will use a computer with a Windows 10 system.
Fill the excel template that we can find go to step #1 filled with the name VariablesPlateIncubation.xlsx
VariablesPlateIncubation.xlsx 10m
Upload custom labware to app and robot system
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 1m
Export the variable file to the /data/user_storage folder in the robot.
For more information about sending files to the OT-2 go to step #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 python script downloaded from go to step #3 (I named it Example-PlateIncubation.py) to the OT-App
Example-PlateIncubation.py Result of importing the Python script in 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-PlateIncubation.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 like the following pictures.
Labware and liquid set-up layout
Volumes of the antibiotics needed 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 A1 in the labware Samples Plate 'Plate 2' with Amp (1) 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 as well as the finla plates with only samples of media as we state in the excel. 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 from the plate called "Samples Plate 'Plate 2' with Amp (1) Slot 4" in the second picture.
32m
Retrieve labwares from the OT
5m
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