PCR: SOE, Bridge or Megaprimer?

[John Ladasky]

In article <boardman.3-3010981640170001 at path9.med.ohio-state.edu>,
Cindy Boardman <boardman.3 at osu.edu> wrote:
>Request for help:
>
>I am interested in expressing a chimeric protein and have been trying to
>create the junction between the two DNAs by PCR.  I have tried Bridge, SOE
>and Megapriming PCR and have had no luck.  Any generous and enlightened
>souls out there willing to save me?

	I've made mutant proteins by PCR mutagenesis using the megaprimer
approach.  It should be essentially the same as making a chimera.  In my
experience the megaprimer works much better if it prepared to excess with
a blend of Taq and Pfu polymerase, and agarose gel purified.

	Good luck!

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky