Measuring DNA Supercoiling

Michael G. Tencza tencza at med.unc.edu
Tue Jul 27 10:26:05 EST 1993


I have used a 2D system for measuring topoisomers of hepadnavirus
minichromosomes which has worked well.  Briefly:
1)  Use a 2mm single, round well comb to cast an agarose/TBE midigel.
2)  Run a sample overnight (conditions depending on the size of the
maximally supercoiled species).
3)  Cut the sample lane from gel and melt the unused agarose.
4)  R-position the sample lane at the top of the gel support, oriented 90
degrees from the original position.
5)  Re-cast the melted agarose around the repositioned sample and allow to
set.
6)  Soak the re-cast gel in TBE/0.4uM chlorofquine for approximately 8
hours taking care not to expose the gel to light.
7)  Run the gel in the TBE/0.4uM chloroquine buffer used for soaking
overnight to resolve topoisomers in the second dimension.
8)  Detect and analize topoisomers on gel.

Modifications of this method are plentiful in the literature.  One example
is given below, along with some references for ethidium based methods.

1) Krude, T. and Knippers, R.  MCB.  11(12), pp. 6257-6267, Dec. 1991.
2) Keller, W. PNAS.  72(12), pp. 4876-4880, Dec. 1975.
3) Germond, J.E. etal.  PNAS.  pp.1843-1847, May 1975.

^X



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