PCR inespecific band amplification - (Feb/04/2008 )
I'm trying to amplify a gene using pcr real time taqman probes from roche (UPL). The primers I've chosen seem to be good, because I do get an amplifiying curve (although low absorvance). When I run the samples on an acrylamide gel, I see my desired band of 65nt, but also others of 200 nt aprox. The inespecific band is more or less the same intensity as my desired band. So the primers are inespecific. Anyone knows if there is a method to know how much this inespecific band can interact negatively with my desired product? is the low absorvance due to this? or because maybe my gene is poorly expressed in this tissue?
If I sequence the inespecific band, what can I get with this?
Thank you very much in advance
What temperature are you running it at? running a gradient PCR is a good way to see if the band disappears at higher temperatures!
hope i can help
i'm running it at 60ºC, because it's the established temperature for all UPL probes from roche, this way I can run all assays in the same plate. But I will try it, and post the result