Patent ID: 7510856

Claim:
A method for preparing negatively supercoiled plasmid from unligatable open circular plasmid, the method comprising: (a) providing a plasmid solution from host cells containing plasmid, wherein the plasmid solution comprises unligatable open circular plasmid; (b) reacting in vitro the plasmid solution of (a) with one or more enzymes, such that at least some unligatable open circular plasmid in the plasmid solution is converted to 3â€²-hydroxyl, 5â€²-phosphate nicked plasmid; (c) reacting in vitro the 3â€²-hydroxyl, 5â€²-phosphate nicked plasmid with a DNA ligase, such that at least some 3â€²-hydroxyl, 5â€²-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting in vitro the relaxed covalently closed circular plasmid with a DNA gyrase, such that at least some relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid; and wherein at least one of the enzymes used in reactions (b), (c) or (d) is a purified form of the enzyme.