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Purification of RNA-free plasmid DNA using alkaline extraction followed by ultrogel A2 column chromatography

1985; Elsevier BV; Volume: 148; Issue: 1 Linguagem: Inglês

10.1016/0003-2697(85)90636-0

1096-0309

D. Micard, Marie‐Laure Sobrier, J.L. Couderc, B. Dastugue,

Antibiotic Resistance in Bacteria

A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Birnboim and Doly (1979, Nucl. Acid Res. 7, 1513–1523). Plasmid DNA is then separated from high-molecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, 5′-end labeling, S1 mapping, DNA sequencing, and colony hydridization.