It was observed that with same samples and same primers, one qPCR reaction will show multiple peaks, but the second reaction (plate) will have only one Tm peak. Why is it so?
I assume that you are refering to peaks in the melt curve. It depends on where the peaks are, this could be due to primer dimer formation (normally around 70-75oC) or nonspecific amplification. Do you have an image that you can show?
Dear Joyeeta, to evaluate the peaks from an image will be usefull as recommending by Steve Hawkins. Melt curve analysis is related to the size of amplicon. If primer binds more than one region or unspecific amplification, you can see more bands.
Hi Joyeeta,
as Mert said probably you amplified multiple regions of the genome. prior to do something when you're starting with new primers, you should test them in in-silico PCR just to be sure first they are specific to what you're analyzing, and second just to see of only one amplicon you'll have. try at the NCBI primer bast or in-silico PCR at the UCSC websites.
fred
Probably multiple peak due to non specific amplification or multiple binding site of primers or primer dimer formation...............this is the matter of standardization.
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