PCR primer instability

Robert Brushia ez017144 at peseta.ucdavis.edu
Tue Sep 5 19:25:34 EST 1995


On Thu, 31 Aug 1995, Mark Naylor wrote:

> I have been having problems with PCR primer instability lately. I am 
> using 21-24 bp single stranded DNA oligo directed against kinase 
> inhibitor gene exons (e.g. genomic DNA target) that are inherently 
> GC-rich the primers themselves tend to have unusually high GC contents 
> (60-70% some of them). These primers are all selected using computer 
> software (Oligo, MacVector and Amplify) When we first get the 
> primers and put them in solution they work fine, but after a few days, 
> the amplification signal drops noticably and shortly thereafter, they 
> stop giving signal. We have noticed this on all of our kinase inhibitor 
> primers but not on other primers we are using that are made by the same 
> synthesis lab. I suspect that intraprimer interactions are responsible 
> for this phenomenon. Has anyone else seen anything like this?
> 
> Mark Naylor
> mnaylor at cclink.net.uokhsc.edu
> 
I don't know if it will help or not, but are you putting your primers in 
pure water?.  If some DNA's are put in water that is not buffered they 
are unstable due to autocatalytic degradation (DNA is an Acid).  Try 
looking at the stability of your primers in tris buffer.> > > 



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