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A old discussion thread on site-directed mutagenesis
Posted by: user (IP Hidden, Unregistered user, )
Date: December 6, 2004 03:58PM
Some discussion threads from biowww.net
Hi I am trying to find a way to introduce three new amino acids in a recombinant protein, preferrably by PCR. I thought a technique similar to site-directed mut. might work. Does anyone have experience with that? Rasmus 2002-02-11 05:56:27 rasmus (r_wendelbo@hotmail.com) -------------------------------------------------------------------------------- I am doing a site-mutation too on a gene construct. so far, i can\'t seem to amplify the whole vector DNA using two overlapping primers, instead, I got mainly shorter fragments. Can you tell me a little bit in detail how this method works and what kind of Taq will do? thanks. 2001-09-24 10:01:58 gsu (gsu_bear@yahoo.com) Comment List ------------------------------------------------------------------------ Topic: Author: Time: point site mutagenesis problem April Showers 28.04.2004 09:25 Hi, I've been trying to incorporate several point site mutations into a subunit of DNA that I have. I'm using Stratagene's point site mutagenesis kit and I've done several on a different subunit and it worked fine, but with this one, I've faced many problems. The colonies that grow either have the wildtype DNA, have no plasmid, or I many not even get colonies at all. Does anyone have any suggestions? mutagenesis 11.03.2003 11:06 Hi, I am trying to insert a codon in a 20 kb plasmid by Quick change XL site directed mutagenesis kit but I am unable to optimize the parameters. Can anyone help? Thanks.
Re: A old discussion thread on site-directed mutagenesis
Posted by: rodrigo fuentealba (IP Hidden, Unregistered user, )
Date: December 29, 2004 08:28PM
Hum Mutat. 2002 Oct;20(4):323. One-step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs. Scott SP, Teh A, Peng C, Lavin MF. The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston,4029, Qld, Australia. In vitro mutagenesis of large genes has proven to be difficult for a number of reasons, including the number of steps involved and the instability of large inserts. We describe here a single-step PCR method to introduce mutations into an ataxia-telangiectasia mutated (ATM) gene cDNA construct (20 kb). This involved several modifications of the Stratagene QuikChange Site-Directed Mutagenesis Kit. Four sites implicated in the function of ATM were mutagenised in a 20 kb plasmid, improving upon existing methods capable of mutagenesis in DNA constructs up to 13 kb, while maintaining a high efficiency of mutagenesis (85-100%). This approach makes it possible to introduce multiple mutations into large cDNA for structural/functional studies. Copyright 2002 Wiley-Liss, Inc. PMID: 12325032 [PubMed - indexed for MEDLINE]
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