Site-Directed Mutagenesis Kits
Richard E. Showalter
show at agouron.com
Thu Sep 21 19:21:10 EST 1995
> >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!
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.
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
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