Preparative separation of plasmid and bacterial artificial chromosome DNA by density gradient electrophoresis in the presence of linear polymers

A density gradient apparatus was used to examine the separation of differen t physical forms and sizes of DNA. A gradient of sucrose was used to stabil ize thermal convection during electrophoresis in the column (2.2 cm in diam eter). Linear polymers were added to the density gradient and screened for their ability to separate the supercoiled, nicked circular, and linear form s of the plasmid pBR 322. The influence of different concentrations and mol ecular 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 t he 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 arti ficial chromosome (BAC) DNA. A rapidly moving form, presumably the supercoi led form, was resolved from a large amount of E. coli genomic DNA and from sheared forms of the BAC DNA.