strange contamination in real-time PCR in negative control? - (Feb/01/2006 )
I meeted a strange contamination problem in recent RT-PCR work. I used Syber Green mixes. In my experiments, I set two wells of negative control for each primer sets. It is very strange that I get one clear band for one well and another smear for another well if I run gels for them. The most interesting is that I run four times, the same situation reappeared every time. I mean that one well appear contamination but another is not. It is very strange. I made master mix for them and run them together. Any suggestion is welcomed.
of course, contamination is possible in one of your reagents
but, I would put my money on primer-dimer
what do you see from negative control wells with no primer?
what do your dissociation/melting curves show you about your product?