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PCR gene replacement in pombe

S L Forsburg nospamforsburg at salk.edu
Mon Nov 23 07:08:10 EST 1998

Nick (nowen at bioch.ox.ac.uk) writes:
> I am interested in attempting gene replacement using PCR mediated
> techniques using a URA4 marker gene.
> A while ago someone posted a URL of a nice paper on this but due to a
> system crash I cannot find this information.
> If anyone has the posting/URL for a paper on PCR mediated 
> gene replacement or has any suggestions I would appreciate it greatly

General comments:
It works, but because of the reduced homology, there is a much higher
background.  (It's not that pombe is BAD at homologous integration, it's
just very GOOD at non-homologous integration!)  So you will probably
need to screen a fair number--you can screen by PCR with some careful
primer design.  Also, remember these effects can be very locus specific.

Incidentally, it's ura4+ in pombe--URA4 would be a cerevisiae gene.  :-)


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S L Forsburg, PhD  forsburg at salk.edu
Molecular Biology and Virology Lab          
The Salk Institute, La Jolla CA 

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