Hi, I am running a qPCR using absolute calibration to calculate copy number of my gene of interest. I have problems with the efficiency of the reaction. The r2 is great 0.999, so that part I assume is OK. But the efficiency is 70 or 80 %. I checked the primers, the annealing temperature is ok, in the melting curve I see only one pick, and the non template has a very small one, but when I run a gel, there is no product. So I am asking what can I do to improve my efficiency. Thanks.