Hi there,
I am seeing two peaks in melting curve for qPCR.
My negative control shows me that it's not primer dimer nor genomic DNA.
The peaks are similar in height and they are adjacent to each other.
The expected amplicon size is 239bp. I did a 2-step PCR where I denature the DNA at 95'C for 10 sec and anneal + extend for 45 sec at 58'C.
I think it's due to unspecific binding of the primer; my forward primer has TTTT and my reverse primer has TTTT and CCCC and the region of amplificaiton has lots of the same tetra-repeats.
Should I decrease the anneal + extension time from 45 sec to 30 sec? OR should I do 3 step PCR where the extension temperature is 72'C?