qPCR please help

[Anderle29]

Heya,

I am having problems with my qPCR. I am trying to amplify the humand ADH1A gene. I have primers (do not know the sequence) that seem to work fine amplyfing the gene and they give me a nice melting curve. However, my negative control (primers only) comes up positive:-( I have repeated the experiment so many times now it is really frustrating. I have been really careful handeling the plate, samples, and reagents. I have used new water and primers.

Could it be that the ADH1A gene is structurally difficult to amplify??
I really appreciate any comments.

Thanks for your help.

[Aga]

From what you've said it seems that ADH1A gene in your reactions amplifies just fine and the problem is your negative control.
What kind of qPCR do you perform? Is it based on fluorescent probes (like hybrydization probes that can be used with melting curve analysis)/SYBR Green I/other assay? Do you use any commercial kit for ADH1A or do you prepare your assay on your own (primers, polymerase mix etc.)?
If your negative control seems to be positive then what kind of positive signal do you obtain for it? (This actually is based on the kind of assay you perform - whether it is unspecific probe/probe degradation or primer dimers in SYBR Green I based assay.) What is Cp for your negative control?