In vitro mutagenesis
dje0282 at VMS2.TAMU.EDU
Thu Jun 15 20:58:12 EST 1995
>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
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
Dept. of Plant Pathology and Microbiology
Texas A&M University
College Station, TX 77843-2132
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