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. 2007 Sep 26;35(19):6539–6546. doi: 10.1093/nar/gkm702

Figure 5.

Figure 5.

R.NmeDI activity on plasmids with one or two recognition sequences. Reactions were carried out with 0.15 μM of R.NmeDI and 14.2 nM of supercoiled DNAs at 37°C. Aliquots were withdrawn at various times and the reactions were stopped by the addition of 1 μl of phenol and analyzed on 0.8% TBE agarose gel. pUC18 (A) containing one R.NmeDI site and pBluescript KS II (+) (B) containing two R.NmeDI sites were used as a substrate in the reactions. The top panels show agarose gels of time-course series of enzymatic digestion products taken from the reaction. All forms of plasmid DNA: supercoiled (ccc) (×-×), open circle (oc) (□-□), linear (l) (•-•) are marked by arrows. Additionally two linear products of digestion of pBluescript KS II (+) with the enzyme are marked as L1 and L2 (◦-◦). The graphs show the analysis of the data produced by fitting to a first-order rate equation using MathCAD software. The DNA in the substrate and product bands was determined using Gel/ChemiDoc (BioRad) analyzer and quantified with the accompanying software package (Quantity One). The amounts present in individual bands were expressed as a fraction of the total intensity of DNA as visualized by ethidium bromide staining.