inconsistent PCR results - (Jan/31/2006 )
i heve been getting very inconsistent results for my PCR reaction.
i ve been d same standardised protocol for last so many months but now suddenly it is troublin.
at times i get gud PCR products but the other times either it is no PCR product or low yield product
with too many high molecular weight nonspecific bands and primer dimers sometimes
i ve done the temp standardisation and Mg titration as well . also tried use of 5% DMSO but no use
what about primer degradation? have you tried a new batch of primers?
have any of your other reagents been hanging around a long time and possibly degraded? dNTP's frozen/thawed too many times, pH of your TE has drifted....anything like this?
If not too expensive: use a fresh batch of everything, dNTP's, primers, enzyme, buffers and water. See what happens.
If you're buying your PCR-reagents in big batches: aliquot them so you don't have to thaw and refreeze too often. Apart from that: you're sure your template quality hasn't changed?