TITLE: OT-2 Media dispensing and culture inoculation protocol
AUTHORS: Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno

[DESCRIPTION]
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

[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

[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)

[STEPS]
SECTION: Files Preparation
1. 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

  
  

 

SECTION: After-Running
5. Retrieve labware from the OT
SECTION: Files Preparation
1.1. Fill the template with the corresponding values
SECTION: Files Preparation
1.2. Store it as VariablesPlateIncubation.xlsx

 

SECTION: Run Protocol
3. 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

 

 

SECTION: Example
6. 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.
SECTION: Example
6.1. Fill the excel template that we can find    filled with the name VariablesPlateIncubation.xlsx

  
SECTION: Example
6.6.  Turn the HEPA filter module
SECTION: Example
6.9. Start Run

 

SECTION: Example
6.10. Retrieve labwares from the OT
SECTION: Run Protocol
3.1. 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

  

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

 

 

 

0.28. 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.


 

 

 

 

SECTION: Run Protocol
4. Run Protocol in OT
SECTION: Run Protocol
3.2. 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 of that simulation. Many errors are contemplated already and have a specific message that hints the user what could have gone wrong.


 

 

SECTION: Run Protocol
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.
SECTION: Run Protocol
4.2. Labware position check is performed (if needed)
SECTION: Run Protocol
4.3. Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces

 

SECTION: Run Protocol
4.4. Set the labware and reagents as shown in the OT-App
SECTION: Run Protocol
4.5. 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

 

SECTION: Example
6.2. 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/)

  

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   

  

SECTION: Example
6.3. Export the variable file to the /data/user_storage folder in the robot.

For more information about sending files to the OT-2   

 
SECTION: Example
6.4. Import the python script downloaded from    (I named it Example-PlateIncubation.py) to 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
SECTION: Example
6.5. 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.

 

 

SECTION: Example
6.7. Clean platform of the robot that we are going to perform the protocol
SECTION: Example
6.8. Prepare all reagents and labware in the places the App is showing and take into account the notes in step   
SECTION: Setting the robot
2. 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