help for site directed mutagenesis

Sergio sergioal at bbm1.ucm.es
Fri Feb 23 07:25:25 EST 2001


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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