cloning the wrong way?

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed Feb 16 11:43:44 EST 2000


ahmed.elgedaily at dim.usz.ch wrote:

> 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?

Maybe yes. This happens sometimes, and the only way around it is to use a
vector that has no promoter in front of the mcs.

>
> Shall I use a different cloning system?

Worth a try, but the choice depends of course on what you want to do with
the clone later.


>
> Do I have to sequence the clones to make sure that they are the right
> DNA and to confirm the orientation?
>

ALWAYS!!! Especially when using Taq polymerase which is notorious for
making mistakes.

Frank









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