help for site directed mutagenesis

philip hardwidge hardp3 at mayo.edu
Fri Feb 23 09:16:01 EST 2001


Search medline for "PCR-SOE" and "Larry Pease".  Several papers from about
10 years ago offer facile PCR-based methods for making site-directed
mutants.

-Phil Hardwidge
Mayo Foundation

Sergio (sergioal at bbm1.ucm.es) wrote:
: I performed it by using mutant primers and 2 steps PCR:

: primers  --->
: mutant primers -*->

: gene   ----------------------------->
: PCR1   --->.......<-*-
: PCR2               -*->...........<---

: mix products PCR1 and PCR2 after purifying
: 3 cycles of PCR without primers
: add external primers
: 20-30 cycles of PCR
: final product:

:        ------------*---------------->

: clone it!

: Hope this helps
: Sergio

: Keren wrote:

: > Hi
: >
: > I would like undertaken site directed mutagenesis, but never did.
: >
: > Can somebody tell me if it is very difficult to do, how many times it takes
: > to construct clone, if I could obtain some results.... (it is for my
: > thesis). Could you tell me too if you know a good kit that works, and what
: > material I need?
: > Thank you very much,
: >
: > Keren
: >
: > <http://www.biowww.net/forum/read.php?f=1&i=897&t=897>






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