how to induce site-directed mutation?

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Wed Mar 27 11:30:36 EST 2002


Dear Gaodaxia (are you Greek, Chinese, ???)

> my god , i want to induce mutation in a required base in one gene, but it is
> very difficult, who can help me? 
> 

No, it's not difficult. If the desired mutation is flanked by a convenient 
restriction site, you just need one new primer carrying the mutation and the 
restriction site. Flank the mismatch base by approx 10 to 15 bases in 
amplification direction (you might use the same primer design guidelines as 
you find in the Quick Change Protocol)
Then use Pwo/Pfu for amplification, clone and sequence your PCR product. 

The more well known way is referred to as the Qui(c)k Change Protocol. You 
either can buy the expensive kit from (forgot who sells, I never did) or get 
the reagents by yourself from say NEB (actually, it's just DpnI). Just ask 
Google for the receipe: 
http://ww.google.com/search?hl=en&q=quick+change+mutagenesis
There is also a method using single strand DNA, with M13 (???) phage, you 
might find the receip also with Google or have a look into the 
Sambrook/Maniatis Lab Manual.



i assure i will thank you in a your satisfied
> way!

What do you mean by that?

Regards and good luck :) 

Wo (not god), thus sure if I was allowed to post a suggestion.

-----
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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