Purification of RNA-free plasmid DNA using alkaline extraction followed by Ultrogel A2 column chromatography |
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| Authors: | D Micard M L Sobrier J L Couderc B Dastugue |
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| Institution: | 1. Service de maladies infectieuses, Centre hospitalier de Melun, 2 rue Fréteau de Peny, 77 011, Melun Cedex, France;2. Centre d’épidémiologie et de santé publique des armées (CESPA), 408 rue Jean Queillau, 13 014, Marseille, France;3. Laboratoire de microbiologie, Hôpital d''instruction des armées Bégin, 69 avenue de Paris, 94 160, Saint-Mandé, France;4. Service de maladies infectieuses et tropicales, Hôpital d''instruction des armées Bégin, 69 avenue de Paris, 94 160, Saint-Mandé, France;1. College of Life Sciences, Capital Normal University, 105 Xisanhuanbeilu, Haidian District, Beijing, 100048, China;2. Department of Paleobiology, National Museum of Natural History, Smithsonian Institution, Washington, DC, 20013-7012, USA;1. Division of Clinical Oncology, Department of Medicine, Medical University of Graz, Graz, Austria;2. Research Unit Genetic Epidemiology and Pharmacogenetics, Division of Clinical Oncology, Department of Medicine, Medical University of Graz, Graz, Austria;3. Institute of Pathology, Medical University of Graz, Graz, Austria;4. Department of Orthopaedic Surgery, Medical University of Graz, Graz, Austria;5. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria |
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| Abstract: | 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. |
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