insert-positive blue cols

Martin Kennedy cytogen at chmeds.ac.nz
Sun Jan 24 17:27:22 EST 1993


In article <1993Jan21.090729.79 at ebc.ee>, mspeek at ebc.ee (Mart Speek) writes:
> 
> 
> I would like to know whether anyone has encountered problems
> in traditional blue/white colony or plaque screening procedures
> (beta galactosidase assay) when sorting out (non)recombinant
> clones. My question is :-( Could relatively long inserts (>0.5kb)
> sometimes give rise blue (beta gal+) clones? How is it possible?
> Any theoretical or practical solutions (incl refs) to this 
> particular problem are welcome.
> 
> M. Speek
> 
> mspeek at ebc.ee (ESTONIA)

The blue/white assay is based on disruption of disruption of part of the coding
sequence for lacZ by insertion of the cloned DNA fragment.  Any fragment which
is a multiple of 3 bases long and does not contain one of the stop codons (TAA,
TGA and TAG - I think) can allow formation of a "tripeptide" fusion protein
that may still be enzymatically active.  I've had blue plaques from an insert
that was 300bp long for this reason.  Furthermore, any cloned insert that
contains a start codon inframe with the downstream lacZ open reading frame may
also give rise to "blue" plaques/colonies.  In effect, there can be a continuum
in the colour of recombinant clones, from white through to blue.  If you plate
your putative recombinant on X-gal next to a true wild type lacZ vector, you
may be able to detect a difference in the intensity of the blue colour.

All this sort of stuff is thoroughly described in a number of articles by Jo
Messing, but two of the best are:

Messing, J. (oops - don't know the year) New M13 vectors for cloning . Methods
in Enzymology 101, 20-79   (Everything you ever wanted to know about M13 but
were afraid to ask Bionet)

Messing, J. (1991).  Cloning in M13 phage or how to use biology at its best. 
Gene 100, 3-12    (A delightful and informative historical review of the
development of the M13 and pUC vectors - recommended reading for any student of
molecular biology).


Cheers,

Martin

Martin A Kennedy (E-mail = cytogen at chmeds.ac.nz)
Cytogenetic and Molecular Oncology Unit
Christchurch School of Medicine
Christchurch, New Zealand		



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