Analyzing qPCR (Sybr-green) results from one of my last experiments I found that for few samples there was no amplification curve, however specific melt peaks and sharp bands on the gel were observed. What could be the reason for a lack of amplification signal (curve)? Is it because of low concentration of DNA (I used lysed bacteria as a template, so the concentration of DNA was unknown ) or technical problem with the sensor of the machine (Bio Rad CFX96)?
I would be really grateful to have suggestions from you!
I agree with your thought that perhaps the machine is not working correctly for some wells. However, you should run at least one more experiment before you have a technician service it. I am sure you are extremely cautious, but I would always try to replicate the problem, just in case there was some spill over when you opened the plate or the samples got mixed up. Double check that you have not turned the display off for those wells or some other little problem that could easily happen to anyone.
Presuming that you have used a master mix and all the wells are being used to detect the same PCR product, then the other wells are kind of controls for the reaction in the wells without amplification curves, and I think this would mean the detector must not be working correctly. You have the same melting Tm and the bands on the gels are of the same size and similar intensity in the wells with and without amplification curves, correct?
I hope that this helps you, good luck!
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