help for site-directed mutagensis
(by customer from newsnet.com)
Mon Feb 19 01:06:29 EST 2007
> Hi, Folks
> I have been doing site-directed mutagensis for a while. My vector is
> pcDNA 1.3 of ~5Kb plus 1.5 kb insert. I try to have point mutatation
> in one site. Unfortunately, after sequencing, all i got are the
> templates without any mutatation. I set up negative control, which has
> no primers and been treated with Dpn1, and positive control without
> being treated with Dpn1. Both controls works well, which means my
> transformation works well.
> I use 20 ng template in 25 ul reaction in PCR step. Is it too much for
> 0.5 Dpn1 digestion?
> Any one give me suggestion??
I have used the QuickChange II Site-directed mutagenesis kit from
The manual recommends 10ng of template DNA in a 50ul PCR reaction.
I would also recommend doing a 2hr dpn1 digestion instead of the standard
More information about the Methods