site directed mutagenesis

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Thu Jan 15 12:56:57 EST 1998


Hi all!

Hm... best method... 
is there any *best* ?

Just wanna add a comment:

It depends on what you start from.

Recently, I had a point mutation (deletion) in one on my genes (not 
*MY* genes ;-) and decided to kick it out by Pwo-PCR with two 
complementary primers, carrying the cure and digesting the 
methylated parental plasmid away with DpnI. Seems that it has worked 
:-)). 
Primers for this approach should be around 15 bases on each side of 
the point mutation to be introduced.

Now, I had done it with another approach: Meanwhile I have got some 
flanking primers for doing routine controls by PCRing 
fragments from plasmids. If there's a suitable RE site near the place 
to be mutated, I am able to use only one primer for the mutation by 
amplifying the mutated fragment with a high fidelity polymerase using 
one of these common primers (e.g. T7 or Sp6 promotor). Then 
the mutated insert is cut sharp and ligated in into the plasmid with 
the old gene cut out.

hEppi MuHtaDing!

Wolfgang
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Wolfgang Schechinger         
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
http://www.medizin.uni-tuebingen.de/~wgschech/research.htm
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