Site-Directed Mutagenesis Kits

[Chris Ferrer]

In article <DEy70A.BBI at cf.ac.uk>, Jami Grossfield <grossfield at cf.ac.uk> 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


Hi Jami,

During my previous post-doc at UT Southwestern, I used Promega's Altered
Sites Mutagenesis system (it is now Altered Sites II) with very good
results.  Your DNA of interest is cloned into a phagemid, and ssDNA is
prepared (I recommend that a big batch of ssDNA is made at first since it
can serve as template for all your mutagenesis-it keeps well at -20).  The
mutagenesis is performed on the ssDNA. Promega's secret weapon is the use
of a mutS strain post mutagesis that will not repair missmatched bases. 
The mutagenized DNA is allowed to replicate in this strain before the DNA
is isolated and transformed into JM019.  I usually picked 10-12  JM109
colonies, performed ds DNA sequencing (quick and dirty-nothing fancy) on 4
of the 10-12 transformants, and usually got 2 to all 4 to go my way.  The
only time the mutagenesis failed was due to poor mutagenic oligo design
(the oligo had enough homology to another site on my template).  I highly
recommend the kit.  Promega now has versions of the kit that allow
expression of the mutagenized DNA.

Good luck,

Janet