Figure 2.

Minicircle supercoiling using ethidium bromide intercalation. Following purification from bacterial cells, MC^sp has a superhelical density of −0.062. The inset shows an atomic force microscopy (AFM) image of MC^sp. The images show the expected topological features of a supercoiled minicircle with a superhelical density of approximately −0.06. AFM was performed by deposition of MCs onto APS-functionalized mica in air as described previously.⁵² Lane 2 shows that reaction of MC^sp with vTopo collapses all supercoils into a single relaxed band with lower mobility. Increasing numbers of negative supercoils were introduced using in vitro reaction mixtures containing vTopo and increasing concentrations EtBr as indicated. After extraction of vTopo and the EtBr intercalater, the DNA was isolated and subjected to polyacrylamide gel electrophoresis in the presence of MgCl₂ (shown). The negative linking numbers are indicated.