Perfect-match PCR enhancer

Ted Michelini tedm at darkwing.uoregon.edu
Fri Oct 27 23:28:54 EST 1995


In article <46nahu$d10 at news.unimelb.EDU.AU>, Carolyn Calnan
<c-calnan at petermac.unimelb.edu.au> wrote:

> Does anyone know a home made substitute for "Perfect Match" enhancer?
> I am having problems amplifying a 1.4 kb fragment of the mouse p53 gene. 
> It works on genomic DNA extracted from some cell lines but not for 
> others. The DNA looks fine on agarose gels. It amplifies with iunrelated 
> primers and with p53 primers plus PME, but PME is so expensive !!!
> Any sugestions?

You could try T4 bacteriophage gene 32 protein, its a similar protein to
the PME, although Stratagene will give NO info on their product..is it a
protein? etc.I belive BM carries the T4 protein. Also could try different
polymerases. Are you getting nonspecific smear from the bad reactions? PME
is supposed to destabilize all but the "perfect match" right? If this is
the case then higher anneal, hot start etc could help too. Do you have
more protein in some preps maybe? More protienase K in your DNA isolation
might help too.. Good luck

regards,

Ted Michelini
Institute of Molecular Biology
University of Oregon



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