On Wed, 4 Nov 2009, Liao, Xin-hua (NIH/NIDDK) [F] wrote:
>
> 2.  G418 selection: for overexpression, after transformation for around
> 5 days under G418 selection, I spread single cell on KA plate, and then
> pick single colony to culture in 12-well plate. A lot of them can not
> expand in the well anymore. Again, I suspect some WT cells survive
> through the drug selection. 20ug/ml of G418 is used. Even for the real
> colonies growing up, sometimes only a few of them express target
> protein, others do not express but are G418 resistant. This will make me
> picking a stable overexpression cell line is as difficult as screening a
> knock-out strain!

The V18Tn5 vector delivers cloned transformants growing/developing on
solid media 4-6 days after transfection.  Developmental morphology,
reporter expression patterns and protein overexpression can be assayed
directly on the transformation plates.  Bacterial or fungal contamination
virtually never occurs, even if plates are dropped on the floor.
100% of the clones can be started in liquid medium.

There is no longer any need for more cumbersome traditional procedures.
Full information is in Dictybase.


----------------------------

Prof. Harry K. MacWilliams
Biologiezentrum der Ludwig-Maximilians-Universität
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