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Preparative separaton of plasmid and bacterial artificial chromosome DNA by density gradient electrophoresis in the presence of linear polymers
Author(s) -
Cole Kenneth D.
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150191803
Subject(s) - density gradient , plasmid , dna , polymer , electrophoresis , dna supercoil , chemistry , gel electrophoresis , chromatography , biophysics , biology , biochemistry , dna replication , physics , organic chemistry , quantum mechanics
A density gradient apparatus was used to examine the separation of different physical forms and sizes of DNA. A gradient of sucrose was used to stabilize thermal convection during electrophoresis in the column (2.2 cm in diameter). Linear polymers were added to the density gradient and screened for their ability to separate the supercoiled, nicked circular, and linear forms of the plasmid pBR 322. The influence of different concentrations and molecular weights of the polymers was examined on the separation. Polyethylene oxide with a molecular weight of 5 000 000 and a concentration of 0.2% w/v achieved the best separation results for the different physical forms of the plasmid. The order of separation of the different physical forms of the plasmid were linear (fastest), supercoiled, and nicked circular (slowest). These conditions were also used to separate a preparation of bacterial artificial chromosome (BAC) DNA. A rapidly moving form, presumably the supercoiled form, was resolved from a large amount of E. coli genomic DNA and from sheared forms of the BAC DNA.