I have troubles with my PCR. I want to genotype transgenic animals. I have many troubles with it, like for first shot I got some positives, and with later repeats of PCR all my samples come out as negative, while my positive control stays nicely positive. And this is the thing: it is not enough, that my positive control stay positive only, but the signal from the positive control is increasing in later PCRs. Before I´m setting up the reaction, I usually make a dilution of ALL my samples to have the same concentration for each (like 100 or 75 ng/ul), and then I use 2-3 ul (150-300ng) for my PCR. I do the same with my positive control, so it should have the same concentration at all times. and still I experience an increase in signal (comparing, for example, to the signal of the DNA sizemarker, of which I also add always the same amount to the gel - so it is not the fluctuating level of EtBr, which I also try to add always at the same concentration). Does any of you experienced anything like that? I´m going crazy already with my PCRs
This, of course is not my main trouble, my main trouble is not getting positives , but it is also something annoying that I cannot explain.