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A simple and economical procedure for large-scale plasmid DNA isolation.

With the widespread application of recombinant DNA techniques and cloned gene products, preparation of plasmid DNA has become a common laboratory practice. This usually includes growth of plasmid-containing bacteria, extraction of the bacteria and isolation of plasmid DNA from the extract. In large-scale plasmid DNA isolation, removal of bacterial RNAs and other contaminants in the extract may be achieved by cesium chloride isopycnic centrifugation, which requires an expensive ultracentrifuge facility and maintenance, or by digestion with large amounts of ribonuclease, an undesirable procedure in a laboratory also engaged in RNA research. Here we describe an alternative procedure that requires only commonly available laboratory facility and reagents for plasmid DNA isolation from the crude bacterial extract. This is based on the principle that, in concentrated ammonium sulfate solution, large-size RNAs and other contaminants can be precipitated while tRNAs and other small RNA molecules can bind to Sepharose (1). We have found that, in the same ammonium sulfate solution, the plasmid DNA is soluble and does not bind to Sepharose. In a typical preparative operation, we usually employ the enriched medium method (2) to grow a 250 ml culture of plasmidcontaining bacteria and then the alkaline extraction procedure (2, 3) to obtain a crude plasmid DNA extract from the harvested 4-5 ml of wet packed bacteria. Following isopropanol precipitation, ethanol washing and brief lyophilization or vacuum suction (2, 3), the pellet of the crude extract is suspended in 1.8 ml of TIoE5No00 solution (10 mM TrisCl/5 mM EDTA/100 mM NaCl, pH 7.6) and transferred to a 10 ml Oak Ridge type centrifuge tube. After adding 0.1 ml of 10% sodium dodecyl sulfate and 0.1 ml of 20 mg/ml self-digested pronase, the sample is incubated at 55°C for 2 hours. This solubilizes the fine particulates in the mixture. At this point, 2 ml of 3 M (NH4)2SO4/5mM EDTA solution is added and the mixture is kept in an ice-bath for at least 2 hours or overnight. The sample is then centrifuged at 12,00Oxg (10,000 rpm, Sorvall SS-34 rotor) for 15 min at 0-4°C. If the supernatant is still cloudy, the ice-bath incubation and centrifugation steps should be repeated in another clean centrifuge tube. The 4 ml supernatant is brought to room temperature and applied to a 2 ml Sepharose4B column equilibrated with 1.5 M (NH4)2SO4/5 mM EDTA/10 mM TrisCl pH 7.6 (in a disposable plastic column with a pre-tested Sepharose4B lot having tRNAbinding capacity of 15 mg or more per 2 ml in this solution). The column is eluted with the same solution. The first 8 ml of passthrough effluent is collected and dialyzed at 4°C in 500 ml T1oE5N1OO solution, which is changed every 6 hours for 4 times to ensure the removal of ammonium sulfate. The isolated plasmid DNA in the dialyzed sample is concentrated by ethanol precipitation. Figure 1 illustrates the result regularly obtained by the procedure. The isolated plasmid DNA is of high yield and satisfactory for various experimental purposes; these include restriction enzyme digestion and subsequent ligase or phosphatase-kinase reactions, chemical or enzymatic DNA sequencing, polymerase chain reaction, preparations of labeled molecular probes, in vitro transcription by RNA polymerases, bacterial transformation and DNA transfection of mammalian