Inverse PCR

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Thu Mar 14 00:06:06 EST 1996


There is an article in NAR 23(16) 3343, Chevet et al. describing the
use of TMAC in PCR at 60mM concentrations.  They claim it makes AT as
stable as GC, and annealing depends only on length of the oligo.  It
may be worth a try.  Good luck

			Usual Disclaimers	SVEN


In article <4i1mg0$d4n at oac2.hsc.uth.tmc.edu>, "Helen J. Hathaway, Ph.D." <hhath at odin.mdacc.tmc.edu> writes:
> Any suggestions? I am doing site-directed mutagenesis through a very 
> GC-rich area. Inverse PCR has worked for some mutations, but with one 
> other, I always get a (6 bp) deletion at the jucntion. In another case, 
> I almost always get wild-type clones, and the ones that are mutated also 
> have a deletion nearby. In the latter case, I have a problem with an 
> incredibly stable primer dimer. I use Vent polymerase, have tried 
> varying Mg, formamide, and DMSO, I have tried hot starts, slow 
> cooldowns, etc. I use relatively high annealing temps (the MP of the 
> primers is 79 - 92 degrees C). Any advice would be helpful. Thanks.
> 
> 



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