help for site-directed mutagensis

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Sat Feb 17 20:09:33 EST 2007


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



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