Dear Friends, I'm an Italian student in Medicinal
Chemistry and I work in a Molecular Biology
laboratory in Naples (IIGB by CNR, do you know
it?). I have a strange problem and I' d lik to get
some help.. I'm trying to clone the OLE1 promoter
(300bp; OLE1 is the "delta 9 desaturase" gene in
Saccharomyces cerevisiae) into a YEp352 vector
(5181 bp).
But I never reach any good result! I use the
classical protocols: I digest vector and fragment
with compatible restriction enzimes (BamH I and Sph
I); I precipitate the DNA and I perform a ligase
reaction using T4 ligase keeping the mixture at 14°
overnight; finally I transform the obtained plaslid
in competent E.Coli, prepared with calcium cloride
(I use about 10 microlitres DNa for 200 microlitres
cells). I screen the positive cells on a LB-
ampicillin Xgal plates, for the plasmid contains
the ampicillin resistance and the lacZ gene. But,
after all these procediments, I don't obtain any
result: all the colonies are blue (they contain the
wild type vector) or, if they are wwhite, they
contain something different from my promoter. How
can I do? Where do I get wrong? Thanks Sara
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