Vent PCR conditions

Shahram Mori smori at nmsu.edu
Wed Nov 16 01:42:26 EST 1994


awalley at molbiol.ox.ac.uk wrote:

: Hi,

: I'm trying to switch from ordinary Taq polymerase to a proofreading enzyme in 
: my PCR to reduce a high level of Taq errors I was getting in cloned PCR 
: products. My PCR reaction is 45s at 94, 45s at 61, 20s at 72 for 35 cycles. 
: Are any of these stages too long or is 35 cycles too many? I've reduced 
: enzyme and primer concentrations but I still get three products at 500-1500bp 
: rather than the 327bp product I'm expecting.

: Help!

: Andrew Walley,
: IMM, Oxford, UK 
Sorry for this stupid question but
I am not sure if I understand this problem. Your expected DNA is 327 bp
but you are getting stuff at 500 to 1500bp? This means that your primers
are binding farther out than where you want them to.I thought that your
extension time was too short, but that would give you even higher
processitivity and longer pieces not shorter. What is your procedure in
cloning? 
Also it would be good to reduce the number of cycles from 35 to 25. This
way you can avoid PCRing to a great amount, other contaminants that might
be present.
Cheers,

 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





More information about the Methods mailing list