cloning the wrong way?

ahmed.elgedaily at dim.usz.ch ahmed.elgedaily at dim.usz.ch
Wed Feb 16 10:00:57 EST 2000


Hi all:

I have a cloning problem.
I have a PCR product (990bp) obtained with Taq that I cloned into the
INVITROGEN
vector pCRT7/NT-Topo using the INVITROGEN Topo-cloning system.
The transformation into TOP10F' one shot cells (from the kit) gives more
than 100 colonies on Amp-LB plates (no blue/white selection).
When I pick these colonies, prepare the DNA, cut the insert with BamHI,
I always get the insert in the wrong orientation only!
When I pick the tiny colonies that are on the plate and prepare them
I get the insert in the desired orientation.
But I cannot grow these clones more than once!

Do you have an explanation why this happens?

Is the insert toxic to the bacterial cells?
Shall I use a different cloning system?
Do I have to sequence the clones to make sure that they are the right
DNA and to confirm the orientation?

Any comments are greatly appreciated. Please email me privately as well
to my email address.

Cheers,

Ahmed


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