Technical information sheet no. 12: Plasmid isolation from bacteria: some fast procedures

Abstract

Over the last few decades it has become evident that plasmids exist in virtually all bacterial species. These accessory genetic elements are defined as autonomously replicating extrachromosomal DNA (Novick 1980). Plasmids typically account for only a small fraction of a bacterial genome, generally comprising 1 to ,200 kb. However, extremely large megaplasmids, with sizes far beyond 200 kb, have also been detected in R/riz&rttrr and other bacteria. Plasmids of > SO kb might be designated large plasmids. Those used as tools in molecular genetics are often < 10 kb. The aim of this TIS is to describe some fast methods for small-scale plasmid isolation. The methods described lead to crude cell lysates, of sufficient quality for analytical purposes (i.e. agarose gel electrophoresis and restriction analysis), which can be used for further purification of the DNA. A sample of a few microlitres from such a crude lysate can be separated by agarose gel electrophoresis to see whether or not plasmid DNA is present and how many different plasmid species there are, and to determine plasmid molecular weight(s) and the approximate copy number or amount of plasmid DNA. These methods, which are especially suitable for plasmid screenings, have all been tested at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM). They do not require the use of commercially available columns or reagent kits and are practicable for a normally equipped microbiological laboratory. It must be pointed out that, in some cases, the ideal method of plasmid isolation from a particular strain can only be found out by trial and error.