I am performing real time pcr for ACTB. I am getting a ct value of 17-20 in my samples. However , i am getting a peak in NTC too with a ct value around 33-35. Also the dissociation curve for NTC shows the peak around same tm as the samples. Is this contamination ?
I believe you are seeing a low level of contamination. Ideally, a NTC should come up as NA, or in the high 30s. This plus your Tm data suggests contamination. I'd systematically go through the components in the NTC wells (water, PCR buffer, ect.) and check for contamination. You could also try using different pipettes.
I believe it is contamination. You probably have to replace all the reagent and change to a different lab to set the PCR up.
sounds like DNA contamination in your primer mix or water or qPCR mix.
I would suggest to replace the nuclease free water and primers and run again. Contamination is very likely in this case. And also try to clean up PCR setup bench.
Are you sharing your lab bench with someone who perform the same assays yours?
Even if you do best lab practices in performing RT-PCR while others don't, then it is likely that you will continue to see peaks in your NTC. Sloppy workers in a team can be your constant source of contamination.
Hi, i have a question about my NTC values in qPCR assays:
How much variability there may be in the ct values of the NTC between different runs. That is, in run A I had an NTC of 34 and in run B I had an NTC of 39, is this reliable?
Thanks!
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