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 standard...

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Bibliographic Details
Published in:Nucleic acids research 1989-06, Vol.17 (11), p.4359-4365
Main Authors: Simske, J.S, Scherer, S
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Chromosomes - analysis
Cloning, Molecular - methods
DNA
DNA conformation
DNA, Circular - analysis
DNA, Fungal - analysis
DNA, Superhelical - analysis
Electrophoresis - methods
Electrophoresis, Agar Gel - methods
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
nicked circular dna
Nucleic acids
Plasmids
pulsed field electrophoresis
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
supercoiled dna
Time Factors
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/17.11.4359