Degenerate PCR troubleshooting - (Aug/22/2005 )
I have had trouble trying to get my degenerate PCR to work, have tried dropping temp 5 degrees, varied MgCl2 concn and template amount. Extention time is 2mins, which should be enough for my product, expecting about 1.2kb. Primers using 50pmols per rxn. The most I have got is a very low molecular weight smear at a mgcl2 concn of 5mM, template variation didn't seem to affect smear. i was going to try a second round of PCR with this PCR product, any other suggestions? Using touchdown PCR.
you should tell us what's the template you use , cDNA or genomic DNA?
And what organism -- or at least the GC percentage. Are there introns?
I've had good luck using codehop designed degenerate primers. They work better because amplification is enhanced by better recognition at the 3' end of the primer. There is a free program on the web with some details on their primer design and amplification program.