Hi.
I am performing real-time PCR with 10 different sets of primers. All primers were found in references and I checked them on BLAST and with different primer verification programs. They should be okay.
I have approximately 40 different DNA samples to do the PCR reactions on.
However, the melting curve analysis of the different samples show very different profiles across the different sets of primers.
For a few primer sets, the melting curve analysis show the desired distinct peak for all samples. However, for most of the primers, some of the samples show several peaks (not necessarily primer/dimer) and then some of the samples are just fine, showing one peak. Strangely, it is not the same samples which are good or bad, when comparing melting curves for the different primers. Moreover, there is no correlation between previously measured DNA concentrations and whether the reactions are good or bad.
The most plausible explanation would be different efficiencies for the individual DNA purifications, hence perhaps some samples contain inhibitory compounds etc. But then again, it is strange that for some primers, the DNA is actually okay.
Is there anything to do? I have no way of getting more DNA, so that is not an option. I use SybrGreen mix for the reactions, hence I only add primers (in quite low concentrations) and template DNA.
Would it help to increase the primer concentration or perhaps to dilute the DNA samples?
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