PCR Mutagenesis

Shoumo Bhattacharya sbhattac at mbcrr.harvard.edu
Fri Jul 14 17:02:58 EST 1995


neill gingles <nag3 at le.ac.uk> wrote:
>I was wondering if anyone could give me feedback on PCR mutagenesis. I 

You need to have convenient restriction sites around the site so
that the pcr products are about 3-500nt. I use Pfu polymerase, and very
low concs. (20-50uM) dntp; this reduces artefacts. You need about 12-15
nt on either side of the mismatched site, so that the pcr can be done
at relatively high temperatures, again this reduces artefacts. Use
plenty of plasmid as template; this reduces the number of cycles you
need.  The actual stuff can be done in a day.

If you need more details let me know.
Cheers Shoumo





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