how to induce site-directed mutation?
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:
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
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
More information about the Methods