Site directed mutegenesis

Rich Dudley rdudley+ at pitt.edu
Wed Nov 11 08:17:27 EST 1998


Oskar Fdz-Capetillo wrote:

> Hi there:
>     I am just starting to develop my mutagenesis techniques. I am
> wondering about using the "quick change" Kit from Stratagene. I would
> want to know if anybody has used this kit and how did it work.
>     I would like to know any other site mutagenesis protocols if you
> have tried them succesfully.
>     Hoping to hear about you soon and thank you in advance,
>                 Ana Aguirre

I have used several kits, including the Quick Change kit.  The one you
choose depends on what you want to do, and how you want to do it.  The
Quick-Change kit is excellent for making a single point mutation--it is
very quick, and doesn't require any additional subcloning.  However, it
cannot make multiple mutations in distant places, unless you make them
one at a time.  The pAlter from Promega is good for making several
mutations distant from one another at the same time.  It is a lttle
slower, but always effective, and does require subcloning.  We have used
this to make 9 mutations in a single gene.  The Gene Editor kit is an
in-between arrangement, but you must be in an Amp resistant plasmid for
it to work.  It is good for making several distant mutations
simultaneously, but each plasmid can only be mutated once.

Good luck!

rich


--
--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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