Thanks in advance for this help.
I am working with Reverse transcriptase PCR, with lot of genes. my efficiciency is too low around 75 to 80 % few genes, r2 is good, A2607280 is 1.98, also in the melt curve for serial dilutions of standard, I get a single peak but the orientation of the tip of the peak is different for each dilutions. i dont know what is the problem. me working with SYBR green quantifast kit.
also one more thing, will the Tm of the primer show an effect, because some genes tm is a bit above 62 etc but in this kit acquisiton and extension is done only at 60 degrees.
its a great problem and i will be gratelful if someone could help immediately
thanks a lot