In vitro mutagenesis

[Dan Ebbole]

>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
dje0282 at summa.tamu.edu
Assistant Professor
Dept. of Plant Pathology and Microbiology
Texas A&M University
College Station, TX 77843-2132
USA