Preparing Customized Template

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

Here we attach one excel with several sheets:

1. Template to use in protocol
2. Explanation of each variable
3. Several examples

Template_Variables_PCR.xlsx

Fill the template with the corresponding values

Store it as a comma-separated values file (csv) with the name Variables-PCRs-OT.csv (if the file has another name the python script will not work).

Transferring csv to Robot

Transfer the Variables-PCRs-OT.csv to the directory /data/user_storage of the OT system that we are going to use to perform the protocol.

Here we present a summary of how to transfer the files in 3 OS: Windows, Mac and Ubuntu (applicable to most Linux)

Mac/Linux

We will use the command line with scp to transfer the file Variables-PCRs-OT.csv to the OT system.

We need to perform the following line:

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

Windows

There are several ways to pass files from a windows to a linux (for example with a virtual machine).

Here we will use FileZilla (http://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 the Logon Type to key file, change the user name to root and give the directory were the ot key is. Should look something like this

Then press connect and we will have a conection between our computer and the robot.

After this connection, we should be able to move out Variables-PCRs-OT.csv (in our computer) to the directory /data/user_storage in the robot.

Adding the custom labware

Creation of .json file

The description file can be obtained by describing the labware dimensions in http://labware.opentrons.com/create/

Creating description folder

We need to create for our custom labware a folder with the API name containing the description file (.json) called 1.json

Transfer the description folder to the OT robot

We need to transfer the directory or directories created in Step 3.2

For that we need to introduce the following command (in Linux), for Windows process

#Transfering directory to OT (Linux)
scp -i [ot_key] -r [directory_custom_labware] root@[IP_OT]:/data/labware/v2/custom_definitions/custom_beta

Simulating and getting the output (user instructions)

For more information in how to setup the ssh visit the official OT support blog (http://support.opentrons.com/s/article/Setting-up-SSH-access-to-your-OT-2)

Passing python script

To make sure that the protocol will work with the variables we have introduced we need to run the python script in the robot with the OT simulator.

The last script version can be found at http://github.com/Biocomputation-CBGP/OT2/tree/main/PCRsamplePreparation (name of this file is user's choice)

For that we can but instead of passing the variables file, we pass the python script. In this case we can transfer it to the directory /data/user_storage or we can directly pass it to the directory /root (recommended)

Simulating

We should connect to the robot via ssh

#Connect to Linux based OT via ssh (Linux)
ssh -i [ot_key] root@[Robot_IP]

Then we would move to the directory where the python script is with the cd command

Finally we are going to perform the simulation of the opentrons

#Simulate OT protocol (Linux)
opentrons_simulate -o nothing -e [name_script].py > [name_instruction_file].txt 2>&1

In case that you want to see the instruction file before the next step, you can see the instruction file with cat

#Show content file cmd (Linux)
cat [name_instruction_file].txt

Retrieving output file (user instructions)

To retrieve the output from the OT system you can , replicate it but transfering the file from the OT to your computer

In this file we have 3 or more sections:

1. (Optional) Warnings
2. General Information: General Information: pieces of information that are extracted from variables in the variables file and are only remainders
3. Deck Labware Position: this information will be provided by the OT as well
4. Coldblock Reactive Positions: for each coldblock that is needed (with 12 positions) we have a table with the reactives and their respective volumes

In the case that something went wrong, the variables are not consistent or there is not enough deck place to perform the experiments the instruction file will be sorter and will have a single section, Errors .

Setting Labware

Wipe the surface of the deck with 70% ethanol to clean and desinfect the surfaces

Set the respective labwares in the slots as the OT App and the instruction file set

Set the different reactives in their respective coldblock positions, as stated in the instruction file

Load script in OT-App

Now that we have made sure that the protocol is going to work we import the python script in the OT-App.

Load the script

Protocols > Import > Drag the python script

Select Robot to Perform Script

Click in the protocol > Run Protocol > Choose the OT where the file Variables-PCRs-OT.csv is > Proceed To Setup

After clicking in Proceed to Setup you should obtain the setup of the labware as instructed in the Deck Labware Positions section of the instructions file.

The Labware Setup tab should look similar to the following image

Run Protocol in OT

Make sure the needed calibrations are done

Labware position check is performed (if needed)

Start Run

The procedure that the robot is going to do is mainly divided in 3 parts:

1. Creation of primer sets combining water, primer(s) and polymerase
2. Mixing and distributing the primer set(s)
3. Transfering samples to the corresponding wells of all set of primers

Retrieve labware from the OT

Importing maps from robot

This can be done if in Step 1 the variable Optional Map was set to True

There will be as many maps as source plates with the following structure of names: VariableNameMap_PositionSlot.csv

To retrieve we can and reproduce it with transfering the files to the computer.

They will be in the directory /data/user_storage

We want to perform a sample preparation of 56 colonies and 2 controls distributed in 2 source plates with 2 primer sets (Example 1 in the excel file in Step 1)

We will use a computer with an Ubuntu system connected via USB to the robot

Excel temple filled and exported to csv with the name Variables-PCRs-OT.csv

Variables-PCRs-OT.csv

Run protocol

The final results should look like the following pictures

Retrieve labwares from the OT

Export the variables and source plates maps (for the creation of the final map) csv to /data/user_storage

We have never used the labware stacked_abgenes_pcr_96well_300ul, so we must pass the directory regarding this labware to the robot

Pass the scipt that I have downloaded from http://github.com/Biocomputation-CBGP/OT2/tree/main/PCRsamplePreparation (I name it PCR-example.py)

Connect to OT computer, simulate, exit OT system, store the output in instructions_Example1.txt and importing it to the computer

instructions_Example1.txt

Prepare all reactives and labwares

Initial Labware Setup and Coldblocks setup should look like the following picture

Load to OT-App the PCR-Example1.py > Run > Select robot in which we are going to run the protocol

The window and tabs of the OT-App should look like the following pictures

Wipe OT-2 with 70% ethanol

Load protocol labware as instructed in Step 10.6