Rapid procedure for detection and isolation of large and small plasmids

TLDR

Procedures are described for detecting and isolating plasmids ranging from 2.6 to 350 megadaltons in various bacterial species. The method denatures chromosomal DNA with alkaline SDS at high pH and temperature, removes proteins and debris via phenol‑chloroform extraction, and uses the clarified extract for electrophoretic analysis. The approach reduces or eliminates chromosomal DNA while enabling selective isolation of plasmid DNA suitable for nick translation, restriction analysis, transformation, and cloning.

Abstract

Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transformation, and DNA cloning experiments.