I'm in dilemma about my melting curves so I ask you fine people to tell me if these are dimers/non specific products or not?
Melting curve A: ten fold dilutions series from 10^2 (blue) to 10^4 (green), final primer concentration:100 nM, cDNA was made from 2 ug total RNA, Ta=60 °C.
Melting curve B: same dilution series as above 10^2 (blue) and 10^3 (red), 10^4 dilution serie show no ampliciation, primer final concentration 200 nM, same cDNA as above.
What bothers me about melting curve B are different peaks between replicates. Are these different PCR product? How is that possible if 10^3 is diluted from 10^2 and there all replicates have the same melting temperature? On the other hand difference in melting temperatures between replicates is only 0.3 °C. How big this difference (between melting temperatures) needs to be, to conclude there is nespecific amplification?