Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnost ic laboratories. The 'Miniprep' is a rapid method that utilises a centrifug able column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added t o the growth medium and cell lysis was achieved by incubation in 10 mg of l ysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a s tandard phenol/chloroform method, Miniprep completely eliminated chromosoma l interference in gel electrophoresis but otherwise produced identical plas mid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Ba nd densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same prep aration from 30.4 to 196 relative units; mean percentage increases per trac k between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA ext raction from VGREF within 1 h, making the process more suitable for diagnos tic laboratories.