So, we started using silicone sealing mats on our plates for the PCR diagnostics. They seem to fit well and there is no leakage. The problem is, they often tend to pop out during the PCR run, causing all the water to evaporate. We figured that this happens very early, during the initial activation. Any idea why this might happen? Thank you
Hi, Lara!
Maybe I can help.
If I understood well, you are using a 96 well plate and you place this well into a PCR machine? And after that, you seal it with a silicone mat? And your PCR protocol has a hot-start period where you need to activate the polymerase enzyme?
If everything mentioned above is the case, then I am afraid that using these silicone mats could, unfortunately, be the problem. Especially during the initial activation, where you are holding the samples at a high temperature for a longer time and the pressure is building up inside each well, resulting in the silicone mat popping out.
Additionally, this can be an issue due to the 96 PCR well being too smooth as well. But I wouldn't worry about the PCR plate as it is important that it is smooth so that DNA doesn't stay on it.
There are several ways how to avoid this:
1) Try to buy some other sealing mats to see if they work better with your 96-well plate. Or try buying new mats and plates together from the same company if you already didn't do that.
2) Use a PCR kit and protocol without a hot start, although I wouldn't recommend that as it could potentially affect your primer specificity and lead to primer dimers.
3) Try to buy standard 0,2 mL PCR strips for 8 samples in a row. Something like this:
https://ahn-bio.de/consumables/pcr-products/pcr-strips/
I use such strips for my PCR as I have more control with opening and closing these tubes and using them at once only as much as I need. These strips never disappointed me and never popped out while performing PCR, and I performed PCR with them in 5 different PCR machines that our lab has to offer.
I hope this was of some help.
Best regards,
Domagoj
Hi Domagoj, thank you for your answer :) actually, I am not using hot start, but I still believe that the temperature increase is too sudden, the last time they opened as soon as the temperature reached 95°C.
I will consider your advice, as I've also had positive experiences with strips. Thanks again for a quick answer
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