Primer Design

[at sunmail.lrz-muenchen.de]

Joanne D. Andreadis wrote:
> 
> >
> > This is a reposting because I think the first try didn't get through.  If it
> > did, apologies.
> >
> > Does anyone know of a good reference on primer design for site-directed
> > mutagenesis, or a good PC program and where to get it?
> 
> Hi. I just used the overlapping primer method for site-directed
> mutagenesis. It worked really well, however, if you're using this method
> make sure you have good complementarity on both sides of the mutation(s).
> I had a single mismatch and underestimated the instability this would
> cause. People have different ideas on primers, some say to incorp the
> mismatches on the 5' end, others put it in the middle (with 18-20
> complementary ntds on either side). It really depends a lot on what
> method you're using to create the mutation.

Hi, 
We did a lot of mutagenesis reactions with Promega´s pSelect-vector. We 
always set the mismatch in the middle of the oligo and the oligos beeing 
about 30 ntds long (all over). We have an approx. success of 80% clones 
which are both amp-resistant and mutated in the insert.
Sandra
u7g11at at lrz.uni-muenchen.de