Hi all,
I have a problem with melting curve in one primer out of 16 primers, I have a different peak heights (as shown in the pic.)(the other primers give same height), I know that higher peak indicate more products in polymerase reaction than a lower one, which also means the rate of gene expression in samples. But this primer for reference gene, so the expression supposed to be same in all samples.
How can I solve this problem? Should I change the primer concentration for example? or change annealing temperature?
Thank you
It should be check the wells inside the instrument may have some dust and affect on measurement.
The height of the melt curve is not important.
Yes, it's higher if you have more template, but the key information that you get from qPCR is the Ct value.
If your primer efficiency is 95-105% and your controls all worked, then your experiment worked exactly as it was supposed to.
Could you clarify, what is on the Y-axis? In general, this melt result is no reason to be concerned as the primary purpose of the melt is to show the presence of one peak at the same position which is clearly the case. Maybe pipetting errors.
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