help for site-directed mutagensis

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Sat Feb 17 20:12:52 EST 2007


On Feb 17, 8:09 pm, "peter" <peter.ianak... from gmail.com> wrote:
> On Feb 16, 12:35 pm, shiqian... from gmail.com wrote:
>
>
>
> > 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
>
> Steven,
> Probably 20 ng is too much, but I will sugest something else - next
> time you make mutations to include silent mutation that create (or
> abolish) restriction site in your primer so you can screen your
> colonies before sequencing.
> my2c
> Peter

Here is the website: http://watcut.uwaterloo.ca/watcut/watcut/template.php?act=silent_new



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