Primer Design

Joanne D. Andreadis jandread at
Fri Jun 14 14:48:33 EST 1996

> 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.  

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