>High GC PCR

Paul N. Hengen pnh at SIRIUS.COM
Mon Jun 22 15:44:19 EST 1998


Thomas Weissensteiner wrote:

> 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

The TiBS M&R article on betaine can be found on the web at:
http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/jun97.html

I'm pretty sure the "GC Melt" is nothing more than TMAC. You
should definitely look at the references cited in the article
mentioned above, especially the one in NAR with Ken Fong as
author...note that K. Fong is the founding father of CLONTECH ;-)

Hung, T., Mak, K. and Fong, K. (1990) Nucleic Acids Res. 18,4953

If you're using their standard buffer "X", it could cause problems
if it contains K+. If so, you should change buffers to exclude K+.
The following article describing this is also at my archive site:

@article{Hengen1996Jantibs, 
author = "P. N. Hengen",
title = "Methods and reagents - Cycle sequencing through {GC}--rich
regions", 
journal = "Trends in Biochemical Sciences", 
volume = "21", 
number = "1", 
pages = "33-34",
month = "January", 
year = "1996"}

-- 

-Paul.

Paul N. Hengen, Ph.D.
mailto:pnh at sirius.com
http://www-lecb.ncifcrf.gov/~pnh/



More information about the Methods mailing list