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Hi,
I've designed two S. cerevisiae integration experiments, but I'm
not completely sure they are the best strategies. I
would appreciate any advise.
I have two PCR fragments (for two different integration sites)
with the LexA operators, SPO13 promoter and the
SPO13::URA3 gene. Once integrated, it will be used for
negative/positive YnH selections.
(1) The first integration is into an FY250 strain. I have one
SPO13::URA3 PCR fragment with homology to integrate into the
ura3-52 locus. Also, I have an FY250 strain with a Y1H plasmid for
LexA activation (H tag, under the gal promoter). After
the SPO13::URA3 integration, I will be growing this strain on an
SC/HU- 2% gal/raf plate. I will then loose the Y1H
plasmid.
(2) The second integration will be into an FY 251 strain that has a
plasmid already integrated with the URA3 marker. I've
transformed this strain with the same Y1H plasmid (Lex A activator, H
tag, gal promoter). I will be using this transformed
strain for my SPO13::URA3 integration at the URA3 site. A successful
integration should knockout the URA3 gene and shouldn't
give the URA3 phenotype without activiation. Therefore, I will select
on an SC/H- glc plate with 0.2% 5-FOA (for succesful
knockouts of URA3), then grow those transformants on SC/HU- 2% gal/raf
plates.
I hope this isn't too confusing. Any input would be very appreciated.
Thanks,
Justin
Justin Lorieau
Biophysical Chemist
Department of Chemistry
Columbia University
3000 Broadway Avenue, MC 3162
New York, NY 10027
USA
Phone : (212) 854-0362
Fax : (212) 932-1289
E-mail : JLL2006 at Columbia.edu
ICQ : 136771
MSN : digitalderbs at hotmail.com
Yahoo :digitalderbs
