insert-positive blue cols

Mart Speek mspeek at ebc.ee
Thu Jan 28 08:17:46 EST 1993


Fiberman (wsun at jeeves.ucsd.edu) wrote:

: I have had the same experience.  I tried calling Stratagene,
: asking them where does the alpha complemetation peptide start in
: the bluescript vector and how long is the peptide, but the
: technician did not know anything.  If the peptide is short
: enough, cloning an insert into a site that's at the 3' end of
: the polylinker will not give you any white colonies.  Any one
: out there knows how long is the alpha comlementation peptide?

: -fm

    Dear Dr Fiberman;

I don't quite understand your question. But anyway, all you need
to know is that inserts in beta gal expression vectors are cloned
into mcs located after the first 5 amino acid coding sequence of
lacZ gene (e.g. see NEB catalog 1992, pp 147-148). Any disruption
of the lacZ gene if inserts are cloned into mcs of Bluescript (or any
other similar vector) results, in theory, blue to white phenotype
change. This is a basic principle (for other details see excellent
responces here by Caroline Breitenberger and Martin Kennedy)

Unfortunately however, this rule doesn't apply always. 
Quite frequently (much more frequently than one would expect) inserts
can have a ribosome "landing bad" and an in-frame initiator codon
in their 3' terminal portion which provide alternative initiation
of translation (internal initiation or promiscous initiation common
to bacterial ribosomes) of the beta gal peptide. As you may understand
I'm not talking about 3N rule and/or extra domain of beta gal (refered
above). Some folks are waisting time by picking up and analyzing
white cols/plaques which are insert negative. Thus, I wish to 
ALERT those workers:  DON'T FORGET COLONY/PLAQUE HYBRIDIZATION
when analyzing beta gal expression constructs (hybridization is
pretty much foolproof).

I must confess that at least in part, this was the reason why I raised
the question of blue/white colony screening and isolation.

MANY THANKS for EVERYONE who participated in this forum!!!!!!!!!!!!!!!

Mart (mspeek at ebc.ee                                    ESTONIA) 



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