PCR problem

Peter Horn hornpete at pilot.msu.edu
Thu Jun 13 10:52:57 EST 1996


In article <4pf866$j33 at news.accent.net>, sangers at accent.net (stephane
angers) wrote:

> We are trying to amplify a Dna sequence with a lot of CG and after Pcr
> we can't get any amplification (no band on gel) we thing that the
> polymerase is blocked in some secondary structures that are formed.
> Is there  a way to counter that ?
> Stephane Angers
> sangers at accent.net

Stephane,

We work with HSV which is very problematic for PCR due to high GC content. 
Like the other responses, I also have found that addition of DMSO as a
cosolvent vastly improves PCR yield.  Try PCR reactions with 5 and 10
percent (v/v) final concentration of DMSO.  Bear in mind that you might
have to drop your annealing temps beyond what you would normally consider
optimal, due to the polarity change.  A reference I have claims that
glycerol also can assist high GC PCR yield.  It's effects were much more
variable however (not my personal experience; I've never tried it).  If
you're interested in trying that, they suggest varying glycerol percentage
up to 20%.  Using DMSO I've had excellent success amplifying an HSV
fragment that had a GC percentage over 80%.  Good luck.
-- 
Peter Horn
hornpete at pilot.msu.edu
Dept. of Biochemistry
Michigan State University



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