[Yeast] integration problem with YIp vector

Nicolas Brandes via yeast%40net.bio.net (by nbrandes from umich.edu)
Wed Mar 7 11:19:37 EST 2007


Hi,

I need some help:
I cloned my YFP gene into a pRS304 integrating vector (YIp vector) and tried
to integrate this plasmid into the TRP1-locus on chromosome IV of a W303
strain that has a trp1-1 marker. I got colonies which are prototroph, they
grow on SC-TRP plates. My cell are also green under the microscope, but if I
do colony PCR with primers that bind forward in the chromosome and reverse
in the plasmid the PCR is negative. If I use primers that bind both in the
plasmid the PCR is positive. This should tell me that the integration didn't
work or at least that the plasmid is not integrated at the TRP-locus. Is
that true? Could it be that the plasmid is just transformed into the strain
but not integrated? But it is a YIp vector that shouldn't replicate
autonomously. Any suggestions what to do?

Thanks for help,
Nico

 

 

 

 

---------------------------------------

Nicolas Brandes 
University of Michigan
Department of MCDB
Nat.Sci. Bldg., Room 4041 (Jakob lab)
830 N.University
Ann Arbor, MI 48109

 

 

 

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