DNA cleavage of supercoiled and linear DNA with one or two R.EcoR124I_NT recognition sites.

<p><b>(a)</b> Diagrammatic representation of the DNA substrates used: pUC19 (left) and pTK-neo (right). <b>(b)</b> Restriction assay. The endonuclease was incubated with DNA substrates containing either one or two recognition sites, both supercoiled and linear. Reactions were carried at 37°C and stopped with the addition of 0.5 M EDTA at 1, 5, 15 and 120 minutes. Reactions were run on a 0.8% TBE agarose gel with a 1kb DNA marker (NEB). <b>(c)</b> Densitometry scan of the reaction product of R.EcoR124I_NT incubated with two-site linear DNA. <b>(d)</b> Kinetics of cleavage of supercoiled DNA with two recognition sites (pUC19). Reaction products were analysed on a 0.8% TBE agarose gel. Reactions were carried at 37°C and stopped with the addition of 0.5 M EDTA at the time points shown over the range 10–300 secs. M represents a 1kb DNA marker (NEB). <b>(e)</b> Quantitation of supercoiled DNA (blue), nicked circle (red) and linear DNA (green) over the time-course of the reaction, by analysis of the data shown in (d).