Robert M. Mihalek
mihalek at smtp.anes.upmc.edu
Fri Aug 1 14:36:37 EST 1997
In article <5rso9l$98g_001 at leeds.ac.uk>
BMBLDO at leeds.ac.uk (L.D. Ofner) writes:
> Hi everybody,
> I've been trying to sub clone a full length cDNA sequence (4.5kb) into an
> expression vector for ages now with no luck and it's driving me mad ! There
> are EcoR1 sites at either end of the insert in its original vector which I'm
> using to cut it out (there are no other restriction enzymes that I can use
> for directional sub cloning which won't cut the insert sequence or the
> expression vector in the wrong place). I've performed the necessary
> restriction digest for cutting the insert out and cutting the expression
> vector, purified both from agarose gel using Wizard clean up and
> dephosphorylated the expression vector ready for ligation. I've set up
> numerous ligations with differing ratios of insert:vector (including having
> insert in vast excess) and have had loads of colonies growing on plates. But
> when I prep up the DNA ( usually pick at least 10 colonies) and check by
> restriction digest, none seem to contain insert ! I've checked that the
> vector is dephosphorylated by tranforming my competent cells with it and I
> get no resulting colonies on plates. Help ! Where am I going wrong? I'd be
> grateful for any advice or suggestions (soon before I go totally insane !).
> Thanks. Lisa.
You may want to consider the possibility that the cDNA is being
expressed by the bacterial host and is toxic to the bacterium.
Therefore, your positive subclones are being successfully transformed,
but then the host is killed.
Perhaps cutting the cDNA with a four base recognition enzyme like Alu1,
Bfa1, or Dpn2 and then shotgun cloning the whole mess or gel purifying
each fragment then cloning them all could get you around an
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