Multiple in vitro mutagenesis reactions

Dr. David J. Meyer meyerdj at phibred.com
Tue Nov 16 15:03:38 EST 1999


"Dr. Duncan Clark" <Duncan at nospam.demon.co.uk> wrote:

>Hi Folks,

>We need to do about 50 mutations on a gene. It's too long, expensive and
>problematic to synthesise the gene again from scratch so the idea is to
>do it by multiple in vitro mutagenesis reactions, probably using
>Kunkel's uracil method and a pEMBL like vector. Probably native T7 DNA
>polymerase, T4 DNA ligase, kinased oligos. 

>We've used Kunkel and M13 vectors very successfully in the past but only
>for single mutants. This time I need an expression vector and M13 will
>not do, hence a pEMBL type vector

>So how many mutagenic oligos can one use in a single reaction i.e. if I
>put in 10 in one reaction, what will be the no. that give mutations. If
>I screen 50 colonies will we get one with all 10 mutations or just say 5
>mutations or 2 mutations etc. We will pick the one with the highest no.
>of mutations and use that for the next round of ten and so on until we
>get the lot. I'm just trying to reduce the number of rounds of
>mutagenesis.

>Each oligo will either add or take away an RE site so we have a screen
>for mutants. Screen will be PCR and subsequent RE digestion and gel
>analysis. 

>All ideas welcome.

>Duncan

> 
>-- 
>The problem with being on the cutting edge is that you occasionally get 
>sliced from time to time....

>Duncan Clark
>GeneSys Ltd.
>Tel: +44(0)1252376288
>FAX: +44(0)8701640382
>http://www.dnamp.com
>http://www.genesys.demon.co.uk


I have run 13 at a time before. In a small number of minipreps I found
a couple which had 11/13 mutations.


David J. Meyer, Ph.D.
Quality Traits
Pioneer Hi-Bred International, Inc.
7300 NW 62nd Ave.
Johnston, IA   50131-1004

Ph. 515/254-2639
FAX 515/254-2619
Email: meyerdj at phibred.com





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