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:
>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
>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
>All ideas welcome.
>The problem with being on the cutting edge is that you occasionally get
>sliced from time to time....
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.
Pioneer Hi-Bred International, Inc.
7300 NW 62nd Ave.
Johnston, IA 50131-1004
Email: meyerdj at phibred.com
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