title: Preparation of pure plasmid or cosmid DNA using single-strand affinity matrix and gel-filtration spin columns.
abstract: A rapid method has been developed for ultrapure plasmid or cosmid DNA isolation from ten-mL to several hundred-mL cultures of Escherichia coli (midi to maxi prep). A cleared lysate is prepared by alkaline lysis, followed by a quick alcohol precipitation step. Denatured bacterial DNA and RNA having at least 20 nucleotides of single-stranded regions are removed from the supercoiled plasmid by binding strongly to the single-strand affinity matrix (SSAMTM). Plasmid DNA is then effectively purified on a gel-filtration spin column to remove SSAM, proteins, small RNA and salts. This method produces consistent yields of high-quality plasmids that are suitable for use in many molecular biology applications. In addition, recombinant cosmids of approximately 46 kb can be purified intact, free of chromosomal DNA.
authors.affiliation: CLONTECH Laboratories, Inc., Palo Alto, CA, USA.
authors.forename: T T
authors.initials: TT
authors.lastname: Pham
authors.forename: S
authors.initials: S
authors.lastname: Chillapagari
authors.forename: A R
authors.initials: AR
authors.lastname: Suarez
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids / Q000737:chemistry / Q000235:genetics; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D004262:DNA Restriction Enzymes