Site-Directed Mutagenesis Kits

[Richard E. Showalter]

> >Jami Grossfield wrote:
> 
> Hi- I am trying to do site-directed mutagenesis and thought using a
> >commercially availbale kit would make this easier. I ordered the
> >stratagene ExSite kit- and their positive control doesn't even work! (yes
> >I have tried varying parameters, tech. support, etc.)
> >
> >1) has anyone else had a problem with this kit?
> >2)does anyone know a good kit for deletion and point mutatation
> >site-directed mutagenesis kits? one they have used??
> >
> >I know clontech makes one, and NEB- but I would like input from users
> >before buying another kit! please let me know if anyonw out there has
> >successfully used the ExSite kit from stratagene too!
> >
> >thanks!
> >
> >jami
> 


I would have to second the Bio-Rad Muta-Gene kit.  We have made our
expression vectors phagmids by placing an F1 ori in the plasmid.  This way
you can make a series of mutants in a gene that you can already express
without having to do *any* recloning of the mutant construct, and, you can
do double or single stranded sequencing on the same plasmid to verify the
mutation.  I am surprized to see such a low mutation frequency from the
above poster.  I generaly get >80% mutation frequency.  I only need to
sequence 1-2 isolates per mutation reaction to get the clone I want.  I do
tend to make my mutation oligos long >30bp and I am very careful about the
primer to template ratios as recomended in the protocols.  I have used it
recently to generate over 30 different single and multiple site AA
substitutions, deletions, insertions, restriction site modifications, you
name it.  Works for me.  YMMV.

Regards,

-- 
Richard E. Showalter             "Hey Hey Hey Hey it was
Senior Associate Scientist        the DNA.  Hey Hey Hey Hey
Agouron Pharmaceuticals, Inc.     that made me this way."
show at agouron.com                  Queen-Shear Heart Attack