Opentrons Thermocycler with silicone pad

Opentrons
1

Hey everyone!

We’ve a brand new Opentrons Thermocycler GEN2 which uses a silicone pad to seal the plate during PCR reactions, but we are finding poor performance when we use the silicone pad.

Avoiding the pad using a normal adhesive seal shows results comparable with other brands thermocycler :person_shrugging:
I was wandering if someone has already experienced this using silicone pad with thermocyclers,
thanks!

2

@FLEXpert - any tips/tricks?

3

@fago sorry to hear about the performance issue you’re having. What are the phenotype of the performance issues you’re seeing? If available, quantitative comparison data would be great. "

In the meanwhile I’ll reach out to team members within Opentron on potential solutions & get back to you.

4

Hi @FLEXpert!

The major performance issue we’re facing is poor PCR amplification…

What we’ve done:

  1. first we started implementing the majority of Illumina CovidSeq Assay protocol (it is a NGS library preparation protocol) on OT2 with GEN2 thermocycler; we followed Opentrons guidelines about themocycler seals. Our first run was not comparable with library prepared manually and with Biorad C1000 as thermocycler in terms of DNA quantification during the pool step (as described in CovidSeq protocol).

  2. So we started investigating the cause of that: all robot liquid operations seemed ok, than we concentrated on thermocycler.
    We found two main point:

  3. Thermocycler has a block temperature during PCR cycles of 2.5°C less than Biorad C1000 (which is calibrated and used successfully) - I’m already in contact with OT support team for that.

  4. we tried the thermocycler with “simple” Covid PCR amplification (which is an analysis that we do every day); we divided the plate in two parts: one sealed with standard PCR adhesive seal, the other left to be sealed with themocycler silicone seal. The seal has been cleaned with bleach and water as described in OT documents (see here).
    The DNA quantification (with Qubit HS Dna Assay) results after 44 amplification cyclers are:

Sample OT silicone seal OT adhesive seal Biorad C1000
1 6,17 ng/ul 20,2 ng/ul 17,6 ng/ul
2 5,7 ng/ul 15,3 ng/ul 16,6 ng/ul
  1. So we had another test on the same Covid PCR with a brand new silicone seal without cleaning it with bleach and water and the results are comparable between OT thermocycler and Biorad thermocycler (but it is still out of opentrons raccomandations about cleaning the seal :person_shrugging: )

I don’t know if you see some rough error in our procedure, we tried to follow OT guidelines the best we could…

Thanks!

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5

Thank you for the information @fago. I have found the case referenced, which I confirmed as being escalated by support team. Allow me to follow up with you once resolution is identified.

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6

Having similar issues with the GEN2 thermocycler and silicon pads. Is there a better way to clean the pad? I’ve tried bleach and water, 70% ethanol, water itself (always nuclease free). The first few runs worked really well, then I cleaned the pad and results have been bad. Change the pad and cleaned again, also bad. Nanodrop shows >70% reduced yield and curve. doesn’t look as good as normal.

Is it worth buying adhesive pcr plate seals from thermo to fix the issue?

7

Hey @mRNAgent - I’m going to ask this in our support channel and get back to you. Are you running on an OT-2 or Flex?

8

Hey- can you please email support@opentrons.com and copy meghan.ferzoco@opentrons.com? I will make sure we post their answer here as well for visibility. They want us to log your problem internally.

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