PCR mutagenesis

Steve Minchin S.D.Minchin at bham.ac.uk
Mon Jun 20 09:44:45 EST 1994


In article <2tsoar$n17 at masala.cc.uh.edu> bchs1b at Elroy.UH.EDU (Michael Benedik) writes:
>Path:
>bham!warwick!lyra.csx.cam.ac.uk!pipex!swrinde!news.uh.edu!Elroy.UH.EDU!BCHS1B
>From: bchs1b at Elroy.UH.EDU (Michael Benedik)
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: PCR mutagenesis
>Date: 17 Jun 1994 17:59:55 GMT
>Organization: University of Houston
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>Message-ID: <2tsoar$n17 at masala.cc.uh.edu>
>Reply-To: bchs1b at Elroy.UH.EDU
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>Does someone have the protocol/reference for doing random mutagenesis
>by PCR amplification of a target fragment. I think you just change
>nucleotide concentrations and/or add Mn++, but I don't have the details.

>Thanks.

>----------------------------------------------------------------------
> Michael Benedik                                INTERNET: Benedik at uh.edu
> Dept. of Biochemical & Biophysical Sciences    
> University of Houston                          BITNET: Benedik at uhou
>-----------------------------------------------------------------------

Hi Michael,

I answered a similar query about a week ago. We have used PCR mutagenesis 
quite successfully. We do have a good selection for the desired mutants and 
therefore we normally screen many thousands of colonies.

It is not possible to use just one protocol for mutagenesis of different 
fragments.

There is a good review of PCR conditions and the fidelity of DNA polymerases 
by Eckert and Kunkle in the book:

PCR - A Practical Approach, Edited by McPherson, Quirke and Taylor.
Published by IRL Press at Oxford University Press.

I hope this helps.

All the Best

Steve


---------------------------------------------------------
Dr. Steve Minchin,

School of Biochemistry, The University of Birmingham,
Edgbaston, Birmingham, B15 2TT, U.K.

E-Mail
InterNet:       S.D.Minchin at bham.ac.uk
Janet:          S.D.Minchin at uk.ac.bham

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