Vent PCR Woes

M. L. Huang rin0mxw at bumed30.med.navy.mil
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.
>
>Thanks,
>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 bumed30.med.navy.mil
************************************



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