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2, 0.5 mM DTT, 0.1 mM EDTA and 30 μg BSA, in the presence of the drug at 50 μM (total reaction volume 10 μL). Two units of human DNA topoisomerase II were added to the duplex, preincubated as described, and incubated for 30 min at 30 °C. The DNA-topoisomerase II cleavage complexes were dissociated by addition of SDS (final concentration 0.5%) and of proteinase K (Sigma) to 500 μg/mL, followed by incubation for 30 min at 55 °C. DNA samples were then added to the electrophoresis dye mixture (5 μL) and electrophoresed (35 V/cm) in a 1% agarose gel in TBEx1, containing ethidium bromide (1 μg/mL), at room temperature for 2 h. Gels were washed and photographed under UV light.
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