PCR Sample Prep Automation on a Budget

Since its invention three decades ago, PCR has revolutionized molecular biology in research and clinical laboratories all over the world. This has led to massive advances in basic research, as well as in the diagnosis and classification of infectious diseases, inherited disorders, and cancers, to name a few.

Why Automate PCR Preparation?

Many researchers prepare PCR reactions manually, often relying on multichannel pipettes for increased speed and throughput and to reduce the risk of repetitive strain injury from excessive pipetting. However, small pipetting errors are often inevitable during the transfer of very low volumes. These, together with the potential sample cross contamination that may occur even with the most careful pipetting skills, represent common sources of error that are introduced during PCR preparation.

By automating PCR preparation, you can significantly reduce errors by accurately pipetting volumes down to 1 µL and reducing the total number of pipetting steps required. If you’re not tempted to automate yet, just imagine the hours you can save on repetitive pipetting every week with an automated setup! So, what are you waiting for?

Hundreds of Users are Automating their Sample Prep with the OT-2

The OT-2 from Opentrons is the world’s most affordable high-precision pipetting robot, which uses integrated electronic pipettes to transfer liquids and run experiments for biologists in their labs. These electronic pipettes are a novel design created by Opentrons, providing scientists with fast, accurate, and precise liquid transfers for a fraction of the price of other lab automation systems.

Automated preparation of PCR and qPCR reactions is the most popular single application on the OT-2, with hundreds of users worldwide. The OT-2 offers a simplified workflow that can accommodate throughput in the dozens to hundreds of samples per week range. If this is not enough, you can easily scale up your throughput by just buying another OT-2.

Here, we share our guide for automating PCR preparation on the OT-2.

Getting Started - Preparation is Key

As with all experiments, preparation is critical to a smooth workflow and reliable results. The most effective way to automate PCR preparation on the OT-2 is to us the robot to combine samples (i.e. template) and PCR mastermix in the reaction plate that is destined for the thermal cycler or qPCR instrument. The mastermix should be prepared in advance, ideally on the OT-2 robot for maximum precision. Here are the parameters you should pay attention to as you prepare for automation on the OT-2.

Choose Appropriate Labware

The OT-2 supports various labware for PCR. We recommend full-skirted 96-well PCR plates for easy and secure placement on the robot deck, or if your thermocycler doesn’t support fully-skirted plates, you also have the option of using a 96-well PCR aluminum block to hold semi-skirted plates and PCR 8-strips. Taking into account the span of dimensions across PCR plates and 8-strip formats, we suggest that you find a labware definition in our software that closely matches the labware in your lab. Alternatively, you can use the ‘labware.create’ command to input the exact dimensions of your PCR plate. You can find more details about supported and custom labware on the OT-2 in our Labware Library.

Check Your Pipettes

Make sure your OT-2 is fitted with suitable pipettes for the volumes you will need to work with. The p10, p50 and p300 pipettes in single and multichannel formats can accommodate total reaction volumes for PCR experiments from 10 to 200 µL. Check out our OT-2 Pipette White Paper for more information on our pipettes and pipetting volumes as low as 1 µL (1). If you’re preparing any part of the PCR mastermix by hand, ensure that your pipettes are accurately calibrated.

What it Looks Like: PCR Automation in 3 Steps

STEP 1. Design your experiment

Here, you have a few options:

Option #1: Start from scratch using our intuitive, point-and-click Protocol Designer application to choose your labware, reagent starting locations, and define each step of your protocol.

Option #2: Choose from our pre-made PCR preparation templates on the Protocol Library. If your requirements fit into the constraints of these templates, they can quickly get you up and running with shortcuts to generating the protocol you need.

Option #3: Each purchase of the OT-2 includes a free protocol consultation and one free custom protocol, and we can help you to design this if needed using our open-source Python API.

Option #4: You can also script your own protocol using our open source Python API - we have lots of documentation to help you get started!

STEP 2. Make a super-mastermix

Standard PCR mastermixes contain many components of PCR, such as polymerase, dNTPs and buffer. These reduce the impact of pipetting error and the number of pipetting steps per PCR reaction, as well as reducing reagent waste. A ‘super-mastermix’ that includes the standard mastermix along with the forward and reverse primers minimizes pipetting steps even further, requiring only one tip per PCR reaction. Thus, you should prepare a super-mastermix by combining your forward and reverse primers and PCR mastermix in one reservoir.

Samples and reagents can be kept at 4C using the OT-2’s Temperature Module paired with our aluminum block set, while the robot is running, if needed.

STEP 3. Get ready to run!

Place your samples and reagents on the deck of the OT-2 and perform the the simple calibration process included in the Opentrons Run App.

Then, hit run! This allows the robot to do a bulk dispense step with the super-mastermix into every well, which prevents reagent waste and makes the run more efficient.

Grab a Coffee While the OT-2 Gets to Work!

Once you hit the run button, the OT-2 will start by distributing mastermix into your 96-well reaction plate or PCR 8-strip. Then, it will mix your samples with the mastermix in the reaction plate or PCR 8-strip, potentially saving you and your labmates hours of repetitive pipetting every week! When the robot is done, just take your ready-to-go reactions off the OT-2 and place them in your thermal cycler or qPCR instrument to run.

Is It Really That Easy?

Yes, that's how you can automate PCR preparation on a budget with the OT-2. Reach out to us if you have any questions, or if you’d like to have a free consultation with our applications engineering team to create a custom protocol for you!

References

  1. White Paper: Opentrons. Intro to Opentrons Electronic Pipettes Precision, Accuracy, Design, and Testing Methods.