In vitro mutagenesis
DRHOADS at MERCURY.UARK.EDU
Fri Jun 16 08:19:37 EST 1995
> To: mycology at net.bio.net
> From: dje0282 at VMS2.TAMU.EDU (Dan Ebbole)
> Subject: Re: In vitro mutagenesis
> Date: 15 Jun 1995 18:58:12 -0700
> >I have a general question for the community regarding methods for in
> >vitro mutagenesis. We would like to make conditional lethal mutations
> >in a gene we have cloned. We know the gene is essential so it should be
> >possible to make such mutations. What I would like is a simple random
> >mutagenesis method that has been demonstrated by others that works. Any
> >and all suggestions are welcome. I have thought of using Stratagene's
> >XL1-Red strain for this.
> Greg, have you considered a low fidelity PCR approach such as:
> Arcangioli, B., M. Ghazvini and V. Ribes, Identification of the DNA-binding
> domains of the switch-activating-protein Sap1 from S. pombe by random point
> mutations screening in E. coli. Nucleic Acids Res., 1994. 22: p. 2930-2937.
> This approach uses Mn++ to lower the fidelity of taq polymerase yielding
> point mutations. The degree of mutagenesis can be controlled by the level
> of Mn++.
> I haven't done this yet myself but I have talked to people who have done it
> and they like the method very much.
> Dr. Daniel Ebbole
We described a mutagenesis protocol for producing single and double
mutations using PCR in:
Diaz,JJ; Rhoads,DD; Roufa,DJ (1991): PCR-mediated chemical mutagenesis
of cloned duplex DNAs. BioTechniques 11(2), 204-211.
The method uses a gradient of Nitrous acid treatments of a plasmid
that you then PCR for a few cycles to `fix' the mutations and to
limit the mutations to a particular region. Works good on 3-500 bp
regions and can be tuned to give singles, doubles or higher.
||Doug Rhoads || Dept. of Biological Sciences||
||drhoads at mercury.uark.edu || 601 Science Engineering ||
||drhoads at uafsysb.uark.edu || University of Arkansas ||
||501-575-3251 || Fayetteville, AR 72701 ||
|| My Dogma Just Got Run Over by Someone's Karma ||
More information about the Mycology