Isolation of inc P-2 plasmid DNA from Pseudomonas aeruginosa.

Plasmids of the Inc P-2 group found in Pseudomonas species have a buoyant density between 1.716 and 1.721 g/ml. This makes it possible to resolve them from the P. aeruginosa chromosome in analytical CsCl gradients. DNA of a CAM-OCT::Tn401 plasmid will separate from the P. aeruginosa chromosome in two cycles of centrifugation in CsCl without dye in a vertical rotor. Addition of the AT-specific dye Hoechst 33258 permits quantitative isolation of Inc P-2 plasmid DNA in a single overnight centrifugation. Restriction endonuclease analysis of isolated plasmid DNAs reveals molecular weights in excess of 200 megadaltons for all Inc P-2 plasmids examined. This high molecular weight may explain the difficulty in isolating these plasmids by more conventional methods.