Promotor-lacZ costruct / XGal
reinhard at wst.edvz.sbg.ac.at
Tue Dec 7 14:22:34 EST 1993
Dear netters, please help me!!
I am working with a promotor-lacz construct to search for transcription factors
for a Aminoacyl tRNA-synthetase, or for factors that influence the expression of
"my" aaTS-gene in YEAST. With a GAL1-Promotor-aaTS construct inserted, we did EMS-
mutagenesis, searching for colonies growing on Gal-Medium, not (less) growing on
Glucose. Additionally we have the prom.-lacZ to have a 2nd kind of selection.
Now I have 60 mutants selected with Gal/Glu selection, but now it is too unspecifical
so I need the selection on X-Gal. Said mutants do not switch off the promotor but
rather lead to spectrum of quantitative expression patterns (more or less blue).
As of yet I am not able to design out a test, that is sensitive enough AND also
allows to select many colonies.
My questions: Who works with promotor-lacZ and also have/had problems?
Who knows a relevant test that allows testing many colonies?
Who knows of problems with such a construction (literature)?
Who works with a reporter construct other than lac-Z. (literature)?
What's about measuring the lacZ-RNA?
I promise to write a summary of the relevant answers, if there are a lot!
Reinhard at dgen10.sbg.ac.at
University of Salzburg/Austria
Department of genetics
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