Automated Liquid handling Infection Quantification Using OpenTrons (ALIQUOT)

QVEU | NIH/NIAID — ALIQUOT Repository

This repository contains Python protocols for running portions (or the full workflow) of our assay on Opentrons OT-2 and Opentrons Flex, plus R scripts used for downstream data processing/plots.

The protocols are organized by robot and workflow step:

• Serial dilution
• Fix
• Stain
• Full protocol

Each workflow step includes versions for 1–4 plates.

[Note] These protocols are intended for research use in a controlled laboratory setting. Always verify deck layout, labware, volumes, and pipetting parameters before running on a live robot.

Downloading the Protocols

1) Choose robot

• OT-2
• Flex

2) Choose workflow step

• Serial Dilution
• Fix
• Stain
• Full protocol

3) Choose plate count (1–4 plates)

Inside each workflow step folder you’ll find plate-count-specific subfolders for 1_plate through 4_plates.

4) Download the .py file

Open the folder for your selection and download the Python protocol (.py).

[Tip] Name your downloaded file with date + operator initials (e.g., 2026-02-09_LG_ot2_full_3plates.py) to keep runs traceable.

Running a Protocol in the Opentrons App (OT-2 or Flex)

1) Open the Opentrons App

• Launch the Opentrons App on your computer.

2) Connect to the robot

• Power on robot.

• Ensure robot is on the same network (Ethernet/Wi-Fi as appropriate).

• In the Opentrons App, select your robot from the device list.

[Note] If the robot doesn’t appear, verify network connection and that the robot is fully booted.

3) Upload the protocol

• In the Opentrons App, select Protocols.

• Click Upload and choose the .py protocol you downloaded.

4) Review the deck layout

• The App will display the deck map / labware placements.

Confirm every item matches your physical setup:

• Plates (type + position)

• Reservoirs

• Tip racks

5) Confirm pipettes and calibrations

• Verify installed pipettes match the protocol’s.

• Confirm calibration status:

• Pipette calibration

• Labware offset calibration

• Deck calibration (especially after service/moves)

[Tip] If you recently swapped pipettes or moved the robot, re-check calibration before running anything with plates!!!

6) Start the run

• Load reagents as prompted.

• Click Run.

• Monitor the first ~5 minutes closely.

[Tip] Most avoidable errors happen early (wrong tip rack, wrong reservoir well, plate flipped orientation, etc.).