17] A rapid alkaline extraction method for the isolation of plasmid DNA

Abstract

Publisher Summary Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. In bacteria, they carry genes that specify a variety of host properties. In recent years, naturally occurring plasmids have been modified to produce new plasmids, which are used as cloning vehicles in recombinant DNA research. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype (e.g., resistance to a particular antibiotic), often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required depends upon the intended use. Highly purified material can be prepared by the cleared lysate method, which involves a long period of centrifugation in a dye-CsCl gradient. Less purified plasmid DNA is often satisfactory for recombinant DNA studies, and a large number of shorter and simpler methods have been developed. This chapter describes one such method that uses an alkaline extraction step. It is rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours. The DNA is sufficiently pure to be digestible by restriction enzymes, an important advantage for screening. A preparative version that allows isolation of larger quantities of more highly purified material is also described.