56 A. Gabibov et al.
Fig. 4.7.FLD and gel electrophoresis analysis of scDNA relaxation catalysed by
human topoisomerase 1–3′.(a) The typical FLD kinetic curves of pTM 18 and Blue-
Script plasmids relaxation. (b) The semi-logarithmic plotρt−ρ∞vs time for pTM
18 plasmid. (c). The agarose gel electrophoresis of the BlueScript plasmid relaxation.
Experimental conditions: The FLD kinetic study of sc plasmid DNA relaxation catal-
ysed by topoisomerase I was performed in 200μl of the reaction mixture containing
20 mM Tris–HCl (pH 7.5), 0.5 mM EDTA, 75μgμl15μg(μl)−^1 BSA and 5–10 of sc
plasmid DNA. Without special indications the experiments were performed in 100
mM NaCl. The reactions were initiated by the addition of 0. 45 μg of topoisomerase
I. Ten microlitres samples of the reaction mixture with BlueScript plasmid were
used for electrophoresis analysis of the reaction. The enzyme kinetics was stopped
by the addition of 1μl of 10% SDS in the reaction media. Samples were analysed by
1% agarose gel electrophoresis. Gels were stained with ethidium bromide solution
(1μgml)ml−^1 and visualised under uv light. The experiment was performed at 25◦C
under the following electrophoresis conditions: TAE buffer, voltage: 5 V cm−^1