Multiple in vitro mutagenesis reactions

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Mon Nov 15 12:45:15 EST 1999


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




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