A rapid method for the analysis of plasmid content and copy number in various Streptomycetes grown on agar plates.

The techniques available for the identification of plasmid DNA in gram-positive bacteria such as mycelium-forming Streptomycetes are time consuming. Nearly all methods described require the cultivation of the bacteria in liquid medium (1). Simple plasmid isolation techniques have been developed for gramnegative bacteria (2) but they are not applicable to gram-positive prokaryotes. Here, we present a new rapid and reproducible method by which the bacteria to be lysed can directly be taken from agar plates. For the cell lysis a part of a single colony (1—2 mg) is suspended nearly homogeneously in 20 pi TSE solution (50mM Tris, lOmM NaCl, 5mM EDTA pH 8.0) and gently mixed with 20 y\ L-solution consisting of 25% sucrose, 3% Ficoll 400, 1 Unit RNase A and 1 mg/ml lysozyme (freshly dissolved) in TB electrophoresis buffer (90mM Tris, 90mM boric acid, 2.5mM EDTA pH 8.2). Immediately 30 jtl of the suspension is filled into the slot of a submerged 0.2% SDS-containing agarose gel. Depending on plasmid size and separation desired the gel concentrations may vary from 0.5-2.0% agarose in TB buffer. Complete cell lysis is achieved by electrophoretic transfer of the negatively charged SDS into the wells for 40 min at lV/cm. Electrophoresis is continued for 2 4 h at lOV/cm. Before staining with Etfir the gel is rinsed in water to remove SDS.