You need to make sure that the DNA concentration is concentrated, and you need to make sure that the contamination does not occur

regards

It also occurs in negative samples - like no-template control? Do you use the same reagents (same vials) when repeating?

Do I see it correctly that you only see the zig-zag in the beginning and with later cycles the curves look kind of normal?

Thank you all for your answers/suggestions.

@ Harald - plates are always spinned down as instructed by kit's protocol and always checked for bubbles.

@ Nadine - yes, sometimes this happens also in NTC. I always use same reagents and if I repeat run with same reagents and samples all looks normal. That's way this is bothering me. It occurs randomly and I don't know why. I also check if it is well related, but it's not.

Yes yellow curves are kind of zig-zag at the beginning and then catch up with "normal" curve. However in first picture with purple curve is a different story. This is NTC and you can see that in one replicate it starts to fluorescence very early (at 6-8 cycle) and then gradually increases, however it never reaches the threshold. The second and third replicates were negative as they should be.

@ Hassan - protocol that we used in our lab is very strict about preventing contamination. I don't think that is a result of a contamination, because it happens randomly.

Dear Urska,

could you tell us what kind of samples those are? I remember a former colleague who had weird zig zag things due to sample preparation. He was working on plant samples and randomly the wells would make problems e.g. that the foil on top of the plate in some random samples warped and got leaky.

But if the purple one is a NTC, than this is a major problem as there should be no amplification.

To sum it up: happens just randomly, even with NTC and even when using the same reagents? Are you sure that this is not a software error??? Maybe reinstalling the software or let the qRT machine be checked out?

I agree with Nadine random electronic or some wells do not respond properly.

Dear Nadine,

the samples that I'm working on are choo's (hamster). And yes, you are right it happens randomly, using same reagents. Funny thing is that I also thought that maybe something is wrong with foil or plate. However I never thought it could go wrong with software. qPCR machine is checked once a year during maintenance service. It was checked additionally after this issue, but nothing was found.

Dear Joseph,

it's hard to say that some wells do not respond properly. Because that would mean that this happens always at the same well/position but it doesn't.