Verification of real-time amplification specificity - (Mar/25/2007 )
I’m now using Power SYBR® Green PCR Master Mix to do my real-time PCR assay. I designed primers for genes I’m interested. Primer efficiency is very good, and single peak is found in melting curve analysis. Agarose gel electrophoresis shows one band with right size one the gel. I want to know whether this pair of primers is ready for me to do real time PCR with my samples. Or shall I clone the PCR product for automated DNA sequencing to further verify amplification specificity? My colleague told me that we can not clone PCR product generated from UDG-containing supermixes such as ABI Power SYBR® Green PCR Master Mix, and direct sequencing is the only choice. I'm really confused.
Any help or suggestion is really appreciated!!!!
Well, I would start to do my experiment with that primer pair. I tend to sequence amplicons only when there is considerable doubt about their specifity. But if you really want, you can easily cut the band from the gel, purify it and use it for direct cycle sequencing. But I don't think that it is necessary (well, others may think different about that).