PCR: SOE, Bridge or Megaprimer?

Cindy Boardman boardman.3 at osu.edu
Mon Nov 2 10:59:41 EST 1998


In article <71difc$2ag at pmgm.Stanford.EDU>, jladasky at pmgm.Stanford.EDU
(John Ladasky) wrote:

>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



Thank you for replying. I have tried using excessive amounts of gel
purified Megaprimer, but amplified with Taq only, no Pfu.  However I would
be surprised if this is the reason for my failure.  Im wondering if it
isn't simply the design of my megaprimer that is the problem.  Could you
tell me what size  megaprimers you have used, and how many annealing vs
non-complementary bases did they typically contain. 

Cindy



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