I'm optimizing my qPCR assay, so I ran a qPCR with the same set of primers at different melting temperatures, in order to find the one in which the assay proceeds best.
I tested 54, 56, 58, 60, 62 and 64C for the PCR melting step and I added a melt curve analysis at the end of the qPCR run to test for multiple peaks (dimer primers, multiple amplicons, etc)
I got a single peak in the melt curve analysis for all reactions, however, the exact melting temperature of the amplicon was not the same for all of them, even though the template was the same and the primers were the same.
I got a Tm of about 82.4C for reactions at 54, 56,58 ,62 and 64, and of about 81.8 at 60C.
What could be the reason for such a shift? Is it significant?
I ran a gel and I saw a single band per reaction, with the same migration pattern.