Vent PCR Woes

M. L. Huang rin0mxw at
Wed Jun 15 17:38:12 EST 1994

On Fri, 10 Jun 1994 03:53:46 GMT, 
191502931  <SHIVAKD at Meena.CC.URegina.CA> wrote:

>I am having problems running PCR mutagenesis with NEB's Vent polymerase.
>I have tried running the same reactions with Taq which work perfectly.  I have
>tried varying MgSO4 conc., dNTP conc., and annealing temps all to no avail.  No
>product is produced in positive controls, while the same reactions work fine
>with Taq.  So my primers, template etc. seems to be working fine... what am I
>doing wrong?  I am using the manufacturers suggested conditions and buffers. 
>Also, a paper I found describes problems with Vent digesting unprotected
>primers... is this the case and may it be my problem?  I would appreciate any
>comments or tips for optimizing Vent reaction conditions or whole protocols
>anyone could be bothered to post.
>Dave Shivak
Hi, Dave:
   I had the same problem long time ago when Vent just came out. After 
several failed attempts, I called up NEB. They told me Vent has 3'->5' exo 
activity. So, when the primers or dNTPs run out in the reaction, the PCR 
products start being chewed away. This should hold true in all 
proof-reading enzymes. However, I since have switched to Pfu and have 
minimum problems. No mutations found so far. Good luck!

Mark L. Huang, Ph.D.
Immune Cell Biology Program
Naval Medical Research Institute
Code 61, 8901 Wisconsin Ave.
Bethesda, MD 20889-5607 U. S. A.
Voice: (301) 295-1122 Fax: -6857
E-mail: rin0mxw at

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