site directed mutagenesis
wgschech at med.uni-tuebingen.de
Thu Jan 15 12:56:57 EST 1998
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.
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