I'm performing real time PCR and in the last 3 trys I have this problem.
The flourescence curve start to elevate at cycle 5 for ALL the samples (some of them were differents dilutions). I rich hight levels of fluorecesce, I did not see any product in an agarose gel, and the melting curve is a constant (not change of the fluorescence whit temperature).
I can't undertand why I have flourescence whihout a product. Is not a problem of primer dimer cause some of them had been used previously. Is not also the kit, because I check whit three differents brands. I also use two different primer concentration.
Please help me! I need those results!