Topoisomerase I has two general applications: to relax supercoiled DNA, and to generate samples with defined superhelical density. Generating samples with defined superhelical density is described in Analytical Biochemistry, v.122, p. 253, by Singleton and Wells (1982).
For relaxing supercoiled DNA, use the following protocol:
1. Add 0.5 µg supercoiled PhiX 174 DNA, 50 mM TrisHCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, 30 µg/mL BSA, and 1 unit Topoisomerase I to a final volume of 50 µL.
2. Incubate 30 mins at 37 degrees C.
3. Analyze the treated samples on a 1% agarose gel that does not contain ethidium bromide. Stain with ethidium bromide after electrophoresis is complete.