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. 2000 Sep 1;28(17):3323–3331. doi: 10.1093/nar/28.17.3323

Figure 4.

Figure 4

Kinetics of ligation by vaccinia topoisomerase. Reaction mixtures containing (per 20 µl) 50 mM Tris–HCl (pH 7.5), 0.5 pmol of nicked 3′-pNP/5′-OH DNA substrate and 2.5 pmol of topoisomerase (WT, Y274F or Y274A) were incubated at 37°C. The reactions were initiated by addition of topoisomerase. Aliquots were withdrawn at the times specified and incubated with NaCl to 0.5 M final concentration for 30 s. The reactions were then quenched by adding SDS to 0.5%. The samples analyzed by electrophoresis through a 15% polyacrylamide gel containing 7 M urea in TBE. The extent of ligation [36mer/(18mer + 36mer)] was quantitated by scanning the gel using a FUJIX Bio-Imaging Analyzer and plotted as a function of time.