insert-positive blue cols

Caroline A Breitenberger cbreiten at magnus.acs.ohio-state.edu
Thu Jan 21 10:24:04 EST 1993


My technician made an intersting observation in this regard.  He was
preparing a construct for a yeast knock-out like this:
    appr. 500 bp frag - URA3 - appr. 500 bp frag - pBS
He had a URA3 clone in pBS, used PCR to get the upstream gene
fragment and cloned it in, which worked OK.  Then, he used PCR
to get the downstream fragment, tried to put it in the doubly
digested clone.  No luck!  We sat down and went over the whole
procedure, and the strategy he had used made sense, but he kept
talking about screening *white* colonies.  He had obtained blue
colonies in the last step, but since he had not expected any, he
assumed they were contaminants (this from a guy who never had
problems with contamination).  Sure enough, when he looked at blue
colonies, they all had the desired insert.  (The white colonies he
screened were either vector that had only been cut with one enzyme
or aberrant religation of "incompatible"ends.)
In this case, the total insert was about 2 kb.  I did not check the
sequence, but I assume that putting in the downstream fragment
introduced a Shine/Dalgarno and AUG in the right reading frame to
read into the beta-gal gene.  (If you look at the sequences of
many of these lacZ cloning vectors, it seems pretty clear that the
sequence upstream of the cloning site is not necessary for beta-gal
function.)

In another case, a graduate student who is using the Clontech TA cloning
kit (:-)) noted that clones containing his desired PCR-derived insert
(appr. 250 bp) exhibit a pale blue phenotype.  In this case, I think
that the insert provides a continuous open reading frame which results
in expression of the alpha-complementation peptide with an N-terminal
extension.  The pale blue color suggests that this peptide is less
efficient than that of the parental vector.  (The opposite orientation
of the insert presumably gives white colonies - I forgot to ask him.)

Caroline Breitenberger, Department of Biochemistry
Ohio State University, 484 W. 12th Ave., Columbus, OH 43210, USA
614-292-9473.......caroline+ at osu.edu



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