DNA Quality and Transfections - does it really make a difference?

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Wed Feb 24 21:17:35 EST 1999


In article <w.ingram-2502990923380001 at b-wainwright.cmcb.uq.edu.au>,
w.ingram at cmcb.uq.edu.au (Wendy Ingram) wrote:

> I recently had a discussion with a colleague who said that in their lab
> all the plasmid maxipreps used for mammalian cell transfections were
> further purified with cesium chloride prior to use. In our lab we have
> just been using plasmid straight out of Geomed Jetstar maxi kits (or
> occassionally nucleobond kits) for our transfections.
> 
> What I am keen to hear opinons on is whether additional purification steps
> are actually worth the added effort? Do transfection efficiencies increase
> significantly over DNA straight out of commercial maxi kits???  If
> purification really helps then what is the best way to do it?
> 
> thanks,
>           Wendy Ingram.

Other considerations are the method used for the transfection
and the inherent "transfectability" (?competence) of the cells.
     Also, you have to define "efficiency".  I rate this as
the proportion of cells transfected (eg. by X-gal or GFP etc.)
but many comparisons use enzyme activity - you can have a lower
proportion of cells transfected but (transiently) expressing
more protein.

For CaPO4 the cleaner the DNA the better.  The only side-by-side
I did yielded double CsCl > CsCl > QIAGEN = Promega Magic.
You can tell the difference by how nice the precipitate looks.

For lipofection (I've used Lipofectin and LipofectAMINE)
the DNA doesn't seem to have to be as pure for good
efficiencies.  The cells also don't have to be in quite
as good condition as for CaPO4.

I have little experience with DEAE/dextran and electroporation
but have heard colleagues get reasonable efficiencies with
cleaned up (eg. LiCl, PEG) miniprep DNA with electroporation.

If the cells are inherently easily transfectable (COS, CHO etc.)
they are more tolerant of dirty DNA than more problematic
lines (eg. derived from liver cells).

     I hope that explains the depth of my ignorance,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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