Supercoiled DNA/transfection

Darren A. Natale dnatale at box-d.nih.gov
Mon Aug 18 08:19:54 EST 1997


Victor Levenson wrote:
> 
> "Jean-Claude irminger" <irminger at cmu.unige.ch> wrote:
> 
> >Is it possible to transfect cells with relaxed circles or is it important to
> >transfect with supercoiled DNA? Is it method dependent? Is there an easy way
> >to fix relaxed circles to obtain supercoil DNA?

> To re-introduce supercoiles one can try EtBr+topoI with subsequent
> removal of EtBr; one problem with that though is that the supercoiling
> will be positive instead of negative.

Actually, the supercoils will be negative using this method (as well as
if
one were to ligate in the presence of EtBr).  The reason is this:  EtBr,
upon intercalation into the helix, changes the helical repeat (number of
bases per turn) temporarily, and in effect removes negative supercoils
or
introduces positive supercoils.  The topo I will reduce the supercoils
to
zero under these artificial conditions.  When the EtBr is removed, the 
helical repeat assumes its natural value (usually about 10.5 bases per
turn),
and the molecule will again become supercoiled.  Since the introduction
of 
EtBr originally REMOVED negative supercoils, taking it away will again
ADD 
negative supercoils.

D. Natale
dnatale at box-d.nih.gov



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