>High GC PCR

[Mr. T. Weissensteiner]

Jeremy M. Vanderweele wrote Fri Jun 19:
> I'm having a lot of difficulty amplifying a high GC (82-90%) region using
> the GC-melt kit from Clontech.  The region is also pretty repetitive,
> which makes it difficult to get good primers.  Does anyone have a better
> protocol to PCR high GC regions?  (Cheaper would also be nice--Clontech's
> enzyme costs a fortune.)

"GC melt" is probably the same as betaine (use hydrate or free base, avoid 
the HCl complexed preparations!!). I am using a 5.0 M stocks, prepared in 
dH2O and store at -10C (maximal concentration is 5.6 M). Optimal amounts 
in a PCR reaction depend on template, primers, and the final concentration 
of salts. In your case, it is likely to be between 1.2 - 2.0 M but you will 
need to perform a titration

For a review, see
    P.N. Hengen. 1997. Methods and reagents - Optimizing multiplex 
    and LA-PCR with betaine
    Trends in Biochemical Sciences 22, pp.225-226

Regards


Thomas Weissensteiner
Autoimmunity Group
Jenner Institute for Vaccine Research
Compton / Newbury
Berkshire RG20 7NN

T    :  0044 1635 577920
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email : tweissen at RETURNMEhgmp.mrc.ac.uk
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