A rapid alkaline extraction procedure for screening recombinant plasmid DNA H.C. Birnboim and J. Doly A simple method for extracting plasmid DNA from bacterial cells is described. The method allows the analysis of 100 or more clones per day by gel electrophoresis and yields plasmid DNA that is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is achieved without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method. Bacterial plasmid DNAs are widely used as cloning vehicles in recombinant DNA research. After new plasmids are constructed, they may be isolated and characterized with respect to their size and restriction enzyme pattern by gel electrophoresis. One method for preparing plasmid DNA in a highly purified form involves gently lysing bacterial cells, centrifugation to remove the bulk of the chromosomal DNA, and then banding of residual DNA in a cesium chloride gradient in the presence of ethidium bromide. Covalently closed circular (CCC) DNA binds a different amount of the dye than does open circular (OC) or linear DNA and is readily separated from the latter two forms of DNA. A second, more rapid, method for preparing plasmid DNA employs hydroxyapatite chromatography. However, for many purposes including analysis by gel electrophoresis, less purified DNA can be used. Under favorable conditions, a colony of cells can be lysed and plasmid DNA can be detected in crude extracts after electrophoresis. This permits the analysis of many clones and makes screening possible. In this report, we describe another method for plasmid DNA extraction which is both simple enough to permit screening of many small samples, yet yields plasmid DNA in a form sufficiently pure to be digestible by restriction enzymes or to be used to transform other cells. The procedure described is more versatile than other rapid extraction methods. Plasmid DNA can be detected in as little as 0.1 ml of non-amplified liquid culture or in a colony of cells scraped from a plate. The principle of the alkaline extraction method is based on the fact that linear DNA denatures at a narrow range of pH (about 12.0-12.5) while covalently closed circular (CCC) DNA remains double-stranded. This property can be used for purifying CCC-DNA. Plasmid-containing cells are treated with lysozyme to weaken the cell wall and then lysed completely with sodium dodecyl sulfate (SDS) and NaOH.A rapid alkaline extraction procedure for screening recombinant plasmid DNA H.C. Birnboim and J. Doly A simple method for extracting plasmid DNA from bacterial cells is described. The method allows the analysis of 100 or more clones per day by gel electrophoresis and yields plasmid DNA that is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is achieved without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method. Bacterial plasmid DNAs are widely used as cloning vehicles in recombinant DNA research. After new plasmids are constructed, they may be isolated and characterized with respect to their size and restriction enzyme pattern by gel electrophoresis. One method for preparing plasmid DNA in a highly purified form involves gently lysing bacterial cells, centrifugation to remove the bulk of the chromosomal DNA, and then banding of residual DNA in a cesium chloride gradient in the presence of ethidium bromide. Covalently closed circular (CCC) DNA binds a different amount of the dye than does open circular (OC) or linear DNA and is readily separated from the latter two forms of DNA. A second, more rapid, method for preparing plasmid DNA employs hydroxyapatite chromatography. However, for many purposes including analysis by gel electrophoresis, less purified DNA can be used. Under favorable conditions, a colony of cells can be lysed and plasmid DNA can be detected in crude extracts after electrophoresis. This permits the analysis of many clones and makes screening possible. In this report, we describe another method for plasmid DNA extraction which is both simple enough to permit screening of many small samples, yet yields plasmid DNA in a form sufficiently pure to be digestible by restriction enzymes or to be used to transform other cells. The procedure described is more versatile than other rapid extraction methods. Plasmid DNA can be detected in as little as 0.1 ml of non-amplified liquid culture or in a colony of cells scraped from a plate. The principle of the alkaline extraction method is based on the fact that linear DNA denatures at a narrow range of pH (about 12.0-12.5) while covalently closed circular (CCC) DNA remains double-stranded. This property can be used for purifying CCC-DNA. Plasmid-containing cells are treated with lysozyme to weaken the cell wall and then lysed completely with sodium dodecyl sulfate (SDS) and NaOH.