Help me, please!!!! (successive transfections)

Jack Owicki jack_owicki at MOLDEV.COM
Wed Nov 29 14:06:29 EST 1995

If I understand correctly, you want to use puromycin in the second
transfection as a selective agent on cells that have already acquired
puromycin resistance in the first transfection.  I would have thought that
you needed to use a different selective agent for the second transfection,
both to obtain effective selection and to assure long-term maintenance of
both genes that you want to express.  We've been wondering about such
multiple transfections and would also appreciate comments from the net on
the subject.  Of course, this should be distinguished from the usual
(simultaneous) co-transfection procedure, which would be fine for some
purposes, but not all.

Jack Owicki
Molecular Devices
>Hi netters,
>I'm trying to (super)-transfect BHK-cells with an expression plasmid (the
>selection marker is
>Puromycin), which is used in the same cells in other transfection
>procedere before. So I'll
>use sucessive the same expression vector for two different cDNA's.
>Positive clones of the
>first transfection are used as recipient cells for the second one. The
>problem is the
>puromycin concentration during the selection in the second transfection
>step.Are there any
>experiences with this kind of double transfections? The transfections are
>compounded with
>Lipofectin reagent.

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