Premature Fluorescence Increase - (Jul/10/2006 )
I'm hoping that someone here can help me. I've only started doing RT-PCR in the last couple of years, and now I am trying to measure some genes from Brain! I started optimizing the primers to Beta-actin and GADPH. For both genes, in my first run, I did a basic protocol (400nM primers/2uL of cDNA [diluted 1:50] and 10uL of Applied Mastermix; final vol.=20uL). I did this experiment 3 times more and get the same result. An increase in the fluorescence singnal from cycle-1 (vide picture attached) until a exponential increase occurs later. As you can see, Rn of fluorescence is small with small peak in the melting curve also. Someone have any ideia what is going wrong with my qPCR?
I wouldn't bet on it, but either the temperature is wrong, or the primers are crap. Try another ones or raise temperature. The curve looks bad. Are the reagents all right?