An Easy Method to Non-Destructively Separate the Strands of a Circular Duplex Plasmid Chromosome: A Preliminary Report

A straightforward and cost-effective method has been developed for the non-destructive separation of the two strands of a circular duplex plasmid chromosome. This method was adapted from the original protocol reported by Wu and Wu (1996), who achieved strand separation using prolonged low-current electrophoresis. In contrast, the method described here utilizes a two-step denaturation process: initial alkali denaturation to pH 13 to generate “Form IV” DNA, followed by denaturation in 90% formamide at 80°C under low-salt conditions. The resulting single strands were observed to migrate as two discrete bands on agarose gel electrophoresis, indicating successful strand separation without degradation. This approach offers a reproducible, rapid, and inexpensive alternative for investigating the topology and helicity of circular DNA molecules.