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I'm trying to do realtime PCR on a plasmid and I have my positive and negative controls close to each other along with a no-template control. I add 1ul of my template last into the 96 well plates (on the side wall of the well, to be precise) and I seal the plate with optical adhesive seal (applying pressure with hand) and centrifuge afterwards.

While sealing I have noticed that the template drop I add on the side of the wells is being wicked up and is likely spreading into the neighboring wells. I notice amplification even in wells where I added water instead of the template

Has anybody notice the same or has any suggestions for how to avoid this? Do you add template into the liquid in the well or onto the side wall?

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I can't say this happened to me before (but also don't do qPCR all the time).

In principle it should be fine to add the template to the side of the well, since this also lets you visually confirm that you did add the template. However, if you are having problems do consider adding the template to the liquid.

As another way to avoid this problem I would try to first put the adhesive foil lightly on top and then push it down/fix it with something sturdy that has a straight edge (there are special plastic things for this, but a tip-box can also work).

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  • $\begingroup$ Thanks for the answer. I found a plastic applicator to seal plates in the lab. I will try it and see of it improves the result. $\endgroup$ – Prashant Bharadwaj Sep 4 '18 at 5:52
  • $\begingroup$ I have tried now sealing with a straight edge and it definitely does not wick the samples or cross-contaminate them. Thanks for the advice! $\endgroup$ – Prashant Bharadwaj Oct 4 '18 at 20:29

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