Stable transfections....

[Dr. Robert Gay]

Dear Protocolists

We're interested in optimizing the preparation of plasmid DNA for stable
transfection in CHO cells.

DNA quality and the resulting TRANSIENT transfection efficiencies are
greatly enhanced with the use of anion exchange column for the
Midi/Maxiprep DNA preparation steps (we're using Qiagen QIAFilter columns).

That's all well and good but what about for stable incorporation of the DNA ?

Is there a prefered method for recovering the DNA, from the restriction
enzyme digest which is performed to linearise the plasmid at a site of
choice, whilst not compromising the DNA quality ?

Alternatively is it just better to transfect with supercoiled DNA and let
the cells themselves linearise the plasmid - even though a percentage will
be linearised in the gene/promoter of interest.

Hope someone can help !

Thanks in advance

Robert





Dr Robert Gay

Post Doctoral Research Fellow
CRC for Biopharmaceutical Research
Department of Biotechnology
University of New South Wales
Sydney, NSW 2052, Australia

Tel : +61(0)2-9385-1892
Fax : +61(0)2-9313-6710