help for site-directed mutagensis

[shiqiannju from gmail.com]

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??


thanks

steven