From: aidong
Date: 4 February 2012 09:08
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
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