PCR COLONY SCREENING -- HELP

G Gallagher gqva12 at udcf.gla.ac.uk
Wed Jan 4 04:23:47 EST 1995


Help!
we have encountered a peculiar problem when screening "recombinant" 
transformants by PCR. The cells (Stratagene XL-1 blue, MF'kan) are 
transformed by a PCRed up 1.2kb fragment of human TGFbeta1 in a
CMV promoter vector containing Amp and Neo resistance.

Colonies arescreened for recombinants by PCRing with the same primers
used to make the insert. SOme are positive, some are negative, as you would 
expect and the positives are grown up for minipreps.

The plasmid DNA (Stratagene CLearcut) is linearised but always turns out to
be the same size as the parent vector; cutting out the insert gives nothing;
re-PCRing the plasmid shows a negative.

WHAT IS GOING ON????

MAny thanks
Grant Gallagher
Email gqva12 at udcf.gla.ac.uk
fax -44-141-552-3229
phone -44-141-552-3535 x 5441



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