Hello, we are performing primer optimization (annealing temperature) with gradient temperature. For some primers, We obtain single peak but there were variations of the height and melt peak position. Why this is happening? Does anyone have recommendation for this issue?
Thankyou
Thankyou
Hello Dyah Retno Annisa
When you attain a single peak with a primer pair, it means that it anneals to a single site on your DNA yielding a single amplicon (of 1 size). But when you record multiple peaks with a primer pair, it translates to the fact that your primers are annealing to some other targets and leading to non-specific amplification. You can try to play with the annealing temperature, in this case, try diff temperatures (ones closer to the Tm of the primer pair) to reduce the non-specific amplicons. Or else you may have to design fresh primers.
Hi Dyah Retno Annisa
I agree with Jatin Chadha The specificity of the qPCR reaction can be confirmed using melting curve analysis. Your results indicated that your primer is not specific to your target genes, leading to non-specific amplification. It can be eliminated by adjusting the annealing temperature.
In my opinion, you can optimize annealing temperature using PCR standard, use gradient temperature to determine an optimal annealing temperature (2-6 degree lower than annealing temp), make sure you get a single and thick band, the best band indicate the best annealing temp for the amplification of your target genes. Then, you can use it (anneal temp) for qPCR analysis, if you still don't detect single melting curve, you should consider designing new primers.
Break a leg!
Ms. Dyah Retno Annisa ,
The above mentioned are very correct, from your attached picture, I can find that they are not specific, you need to change primer pair for your gene.
Best wishes
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