I am having a set of primers for a gene. I performed qPCR a week before and got anticipated results. But this time with another sample (same species), did in technical replicates each well shows different melting peaks. Even after repeating more than 5 times the problem persists. I did check the sample quality by running qPCR with another primer set and it works perfect. What could be the possible reason?
Hi Murugadas
Your second primers are much better than the first one in terms of producing nonspecific products or forming dimer or cross dimers. The best way to find this out: run a 2% agarose gel with qPCR samples from both primers.
Regards
Hello,
First of all, how long is your amplicon? This is important because of the possibility that several G cluster were present in the amplicon, wich would yield in different joint peaks. On the other hand, if this is sample selective, you should keep in mind the possibility of polymorphism among your samples, that would affect your aligning regions. Also, check your Rx in agarose gel electrophoresis for.the existence of primer-dimer or non specific product amplification. As for dimers, You could correlate the presente of a small band, with a melting peak around 5-10 degrees less than your expected one.
Hi Murugadas
Your second primers are much better than the first one in terms of producing nonspecific products or forming dimer or cross dimers. The best way to find this out: run a 2% agarose gel with qPCR samples from both primers.
Regards
Dear colleagues, which differences in peaks do we consider? How many degrees? In the case of non HRM melting 1 and even 2 degrees defference is normal, but usually with random variation from run to run. I saw it many times on biorad cfx 96. In the case of HRM about 0.5-1 degree is ok.
You can improve it by standartization of starting sample quantity (Quantus, Qbit) and by automatization for example.
Hi Murugadas, Once you ensure your sample source is not having contaminants. Possibly microbiota, or actual contaminants. If your sample is having genetic material from other origin also you may get these type of results.
Have you repeated your experiment with the sample (which gave prompt result) again? If that sample is giving single melting curve, then the problem may be with the second sample.
If by chance the first sample also gives you multiple peaks, then there are chances for the primer contamination.
All the best
regards
Sam
- Badges
- Science method