help for site-directed mutagensis

newsnet customer via methods%40net.bio.net (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??
>
> thanks
>
> steven

I have used the QuickChange II Site-directed mutagenesis kit from 
Stratagene.
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 
1hr.

ST




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