In article <8ejoud$pj9$1 at nnrp1.deja.com>,
sara_t at my-deja.com wrote:
> 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
>> Sent via Deja.com http://www.deja.com/> Before you buy.
>
Are you assuming the blue colonies are negative becasue they are blue, or
did you screen them? If you haven't checked some of the blue colonies for
inserts, then check some anyway (even though they are blue) because
sometimes small inserts don't inactivate beta-gal., so they can still
give rise to plasmids that have make blue colonies.
Nick
--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu
Sent via Deja.com http://www.deja.com/
Before you buy.
