PCR buffers

Dr. S.A.J.R. Aparicio saparici at crc.ac.uk
Thu Sep 3 06:01:24 EST 1992


In article <loryn.5.0 at microbiol.uwa.oz.au>, loryn at microbiol.uwa.oz.au (Loryn Sellner) writes:
> We have recently changed our Taq polymerase supplier, and have run into a 
> few problems deciding what reaction buffer to use. The left over 10x buffer 
> from our old Taq source seems to be giving a lot of background non-specific 
> product when used with the new Taq, which we never saw with the old Taq. 
> When we use the new buffer with the new Taq, we get no background, but we 
> also see a ten fold drop in sensitivity. The major differences between the 
> old and new buffer are that the old buffer contained ammonium sulphate as a 
> salt rather than potassium chloride, and also contained gelatin and 2-
> mercaptoethanol, whereas the new buffer does not have these but does 
> have Triton X-100. I would be interested in hearing:
> 1) any ideas as to why we are getting these results
> 2) anybody else who has encountered problems in using buffers
> 3) anybody else who has their own recipe for making PCR buffer

Loryn, you don't say which suppliers.
I have certainly experienced sensitivity difference in my hands between Cetus Taq and 
Promega Taq. Although we have found the Promega Taq and buffer to be good at times, we have
also experienced sufficient variation so that we no longer use it for sensitive applications.

The buffer we use with Cetus Taq contains only Tris, KCl and MgCl2 - no gelatin, Triton or
anything else, this seems to have given the cleanest and most consistent results.

WE/I have no affiliation at all to any of these companies, so all the usual disclaimers
apply.

yours

Sam Aparicio
MRC Molecular Genetics Unit
Cambridge
CB2 2QH



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