I've been working with qPCR for some months now. I amplify 2 genes (PSEN2 and HN1), and the protocols are optimized for both and have been working fine. The other day I amplified HN1 for some samples and once that finished I amplified PSEN2 for the same samples. The firts qPCR worked fine but the second didn't (no amplification was detected).
I repeated the second qPCR, to rule out errors in the preparation (missing reactives for example) but wouldn't work (no amplification detected in gel electrophoresis neither). Opened new reactives, repeated, and same outcome.
I tried the same reaction in a standard PCR machine (using qPCR reactives) and worked perfectly.
I also tried to amplify HN1 (a qPCR that worked previously) and didn't amplify!
¿What could be the issue?
Information extra:
- qPCR Kit: Rotor- Gene SYBR Green PCR Kit (Qiagen)
- Machine: Rotor gene Q
Thank you
How many freeze thaw cycles have your primers gone through? You may want to consider ordering new primers. I would target this reagent first as it is the only one that is likely different between the two PCR reactions.
Well Ana, you said the same reagents worked in the conventional PCR so the problem couldn't be in the reagents. I would suggest you check your qPCR program and give it a try. Often in our busy schedule, we forget to check the program which we have already run 100 times.
Since your reagents all work, the issue must be with the machine. Make sure no one edited the run conditions. Ask if anyone else has had similar issues too.
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