Pulsed-field gel electrophoresis of circular DNA.
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 Saccharomyces 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 17.8 kb plasmid could be changed. We interpret this result as an increase in the relative mobility of form II in our pulsed-field gel conditions.
Simske
J S
JS
Department of Microbiology, University of Minnesota, Minneapolis 55455.
Scherer
S
S
D002875:Chromosomes / Q000032:analysis; D003001:Cloning, Molecular / Q000379:methods; D004270:DNA, Circular / Q000032:analysis*; D004271:DNA, Fungal / Q000032:analysis*; D004278:DNA, Superhelical / Q000032:analysis; D004586:Electrophoresis / Q000379:methods; D004587:Electrophoresis, Agar Gel / Q000379:methods; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013997:Time Factors
D004270:DNA, Circular; D004271:DNA, Fungal; D004278:DNA, Superhelical