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?
you could run your RT-PCR again and if you see the above mentioned double peaks, then you could run part of your final product on agarose gel. After that if you see two equally-bright bands, you could also use the remaining RT-PCR product for a quick Sanger sequencing. You could also get some new primer pairs to test as well.
p.s. Double peaks can be possible even when primers are specific, think about primers non selective for multiple splicing variants of the same transcript.
you could run your RT-PCR again and if you see the above mentioned double peaks, then you could run part of your final product on agarose gel. After that if you see two equally-bright bands, you could also use the remaining RT-PCR product for a quick Sanger sequencing. You could also get some new primer pairs to test as well.
p.s. Double peaks can be possible even when primers are specific, think about primers non selective for multiple splicing variants of the same transcript.
You can try to raise your annealing temp 3-7 degrees and run your extension at 72. Possible SNP?
If we assume that primers are specifiic, the multiple bands could be due to sequence variation (SNPs) among different samples.
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