title: Two-minute miniprep method for plasmid DNA isolation.
abstract: An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material. Processing time, from E. coli culture to usable plasmid DNA, is two minutes or less per sample. Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number. Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.
authors.affiliation: 5 Prime-->3 Prime, Inc., Boulder, CO 80303.
authors.forename: M C
authors.initials: MC
authors.lastname: Tarczynski
authors.forename: W J
authors.initials: WJ
authors.lastname: Meyer
authors.forename: J J
authors.initials: JJ
authors.lastname: Min
authors.forename: K A
authors.initials: KA
authors.lastname: Wood
authors.forename: R J
authors.initials: RJ
authors.lastname: Hellwig
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014162:Transfection; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes