# Background info and Lens training docs

{% hint style="info" %}
This is for automation bundles only, click here for manual operation:[manual-operation-and-blue-dye-demo-manual-protocol](https://docs.parheliabio.com/product-guides/manual-operation-and-blue-dye-demo-manual-protocol "mention")
{% endhint %}

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The Parhelia Omni-Stainer™ has a standard SBS footprint and is thus compatible with nearly every robotic liquid handler. However, we develop staining protocols for the Opentrons OT-2 robot because of its affordability, accuracy, and programmability.
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#### If you want to get a head start, download our GitHub archive from [here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip)!

## Opentrons OT-2 Robot and Software setup

{% embed url="<https://support.opentrons.com/s/>" %}
^ Click here ^ For Opentrons Set up
{% endembed %}

The Opentrons Help Center at <https://support.opentrons.com/s/> is a fantastic resource that has s[tep-by-step guides](https://support.opentrons.com/s/article/Get-started-Unbox-the-OT-2) as well as [**video guides**](https://support.opentrons.com/s/article/Get-started-Unbox-the-OT-2) **(which we strongly recommend) like this video series below** that takes you from box to protocol.

{% embed url="<https://youtube.com/playlist?list=PLEAtiL9W2-TPpKBBsuIBOIeS0grzHj4jM>" %}

^ This playlist covers ^

1. [Unboxing the OT-2](https://youtu.be/nvjNHod-2hU)&#x20;
2. [Unlocking the OT-2](https://youtu.be/c2fnzlFfCSE)
3. [Setting up the Opentrons App](https://youtu.be/sqkvy8xdPVw)
4. [Attaching Pipettes to the OT-2](https://youtu.be/xmCItodWx2o)
5. [Deck Calibration on the OT-2](https://youtu.be/bKYmFyJACaU)
6. [Tip Length & Pipette Offset Calibration on the OT-2 ](https://youtu.be/7mMtvdcV-dM)
7. [Running a Protocol on the OT-2](https://youtu.be/hiN0EOW3vHM)

{% hint style="info" %}
The Deck Calibration process in video 5 of jogging the pipette tip in the XYZ directions into the center of the engraved "+" symbol on the Opentrons deck is very similar to the labware calibration for the Omni-Stainer™ which is performed using the embossed "+" symbol on the C12 or S12 Omni-Stainer™ lid.\
[As in this video ](https://vimeo.com/779408843)
{% endhint %}

## Checklist

* [ ] Set up Opentrons OT-2 robot. [This set-up guide is a great reference](https://support.opentrons.com/s/ot2-get-started), and our friends at Opentrons are happy to assist you with this if you have any [questions](https://support.opentrons.com/s/information-about-opentrons-support).
* [ ] [Download Opentrons App](https://opentrons.com/ot-app/)
* [ ] [Download Parhelia Bio's base OT-2 scripts and labware definitions from GitHub by clicking here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip)
  * [ ] If you would prefer to visit the GitHub and download individual files our GitHub is at <https://github.com/parheliabio/ot2-scripts>
* [ ] Download customized or stock Omni-stainer script from [Stainworks](https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/)
* [ ] Load protocols and labware definitions into Opentrons software
* [ ] Follow protocol-specific instructions, for preparing and loading your labware and doing protocol setup. We strongly recommend starting with the Blue Dye Demo protocol to get comfortable using the device before conducting actual experiments.

<figure><img src="https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FZpYR9YSQxRTPtOlWYStv%2FScreenshot%202023-05-18%20at%202.44.40%20PM.png?alt=media&#x26;token=09fd7bfc-9147-4442-bd41-a1dde957f360" alt=""><figcaption><p>Example of staining protocol loaded into Opentrons</p></figcaption></figure>

## Step 1 - Place the Omni-Stainer, reagent reservoirs, pipette tip rack, and antibody plate into the dedicated slots on the deck of the liquid handler.

When inserting the labware (plates, reservoirs, Omni-Stainers) into the slot of the liquid handler deck it is important to ensure the complete in-flush alignment of the labware bottom with the surface of the deck. Lack of such alignment may result in tips bending while hitting the bottom of the labware wells. SBS labware should click in or rest fully in a deck location, a common error is one of the back corners not being fully inserted and level with the rest of the labware.

## Step 2 - Create a running protocol using the Staining Works Software

The Python files with staining protocols for an individual experiment can be easily configured using the Staining Works Software. For auditing and accountability we recommend storing the individual day – specific protocols and assigning them a name consisting of the operator name, date, and the purpose of the experiment.

## Step 2 - Start the experiment. Calibration step.

## Step 3 - Start the experiment. Initiate the protocol run.