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Preparative Separation of Plasmid and Bacterial Artificial Chromosome DNA by Density Gradient Electrophoresis in the Presence of Linear Polymers

Published

Author(s)

Kenneth D. Cole

Abstract

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 were 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 sheared forms of the BAC DNA.
Citation
Electrophoresis
Volume
19
Issue
18

Keywords

bacterial artificial chromosome DNA, density gradient, DNA electrophoresis, linear polyers, plasmid DNA

Citation

Cole, K. (1998), Preparative Separation of Plasmid and Bacterial Artificial Chromosome DNA by Density Gradient Electrophoresis in the Presence of Linear Polymers, Electrophoresis (Accessed May 27, 2024)

Issues

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Created December 1, 1998, Updated February 17, 2017