Hi Fred,
While you are working on the optimization of your PCR, I strongly
suggest you to read our paper on partial overlapping primer design
from a link below, which will surely ease your mutagenesis:
http://www.ncbi.nlm.nih.gov/pubmed/21530481
I would be happy to answer your questions should you have.
Cheers
Aidong
On Feb 4, 2012, at 2:14 AM, Fred wrote:
Hi CCP4 list,
Thanks everyone who have answered my post concerning to mutagenesis.
From quick reading most of the answers, the following seems to be a
consensus:
1) Do not concentrate your PCR product;
2) Too much DNA and/or impurities like salts or whatever can inhibits
transformation;
3) Purify your PCR product before transformation if possible or use 3
of 4 microL of it. This is more or less the amount of DNA showed in
the uploaded image.
Kind regards,
Fred
P.S.: I'll let you know the results.
Aidong Han, Ph.D
Department of Biomedical Sciences
School of Life Sciences
Xiamen University
Xiamen, Fujian 361005
China
Phone: 0592-218-8172 (O)
0592-218-8173 (L)
Web: http://life.xmu.edu.cn/adhanlab/