Supercoiled DNA/transfection

Victor Levenson levenson at uic.edu
Fri Aug 15 13:53:40 EST 1997


"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?
>Thanks for your help

Jean-Claude,

After ligation reaction you do not have any supercoiles and yet you
can transform bacteria. 

I do not know about mammalian cells,though there was a series of
papers from Worcell group about relaxation and "re-supercoiling" of
trasnfected plasmids (I believe with SV40 origin); as far as I
remember, they attributed this relaxation-re-supercoiling to stepwise
nucleosome assembly. Later on there were some objections to the whole
experimental setup. Maybe you can find something on relaxed circle
transfection in Worcell's publications.

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. 

Pure curiosity: why do you want to transfect relaxed circles?

Good luck,
Victor Levenson
Res Asst Prof
UIC
levenson at uic.edu



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