Pulsed-field gel electrophoresis of circular DNA

Abstract

Mobility of supercoiled (form I) and nicked circular (form II) plasmid DNAs was determined on two major forms of pulsed-field electrophoresis, CHEF and OFAGE. Plasmids with molecular lengths ranging from 2.30 to 17.8 kilobase pairs (kb) were used with Sac-charomyces cerevisiae chromosomes as standards. Agarose gel concentrations were varied from 0.3 to 2.0 percent, with higher percentage gels resolving forms I and II of smaller plasmids. The pulsing range of 3.7 to 240 seconds resulted in quite variable Saccharomyces chromosomal mobilities on both 0.5 and 1.0 percent gels, while both form I and II of all plasmid DNAs showed relatively constant mobilities with some increase at the shortest pulse times. Using a 30 second pulse time and gel concentrations of at least 1.0 percent, the usual order of migration of plasmid forms for a 17.8kb plasmid could be changed. We interpret this result as an increase in the relative mobility of form II in our pulsed-field gel conditions. © 1989 IRL Press at Oxford University.