title: Autoclave method for rapid preparation of bacterial PCR-template DNA.
abstract: An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.
authors.affiliation: The Center for Environmental Diagnostics and Bioremediation, The University of West Florida, Pensacola, FL, USA.
authors.forename: Keith E
authors.initials: KE
authors.lastname: Simmon
authors.forename: Dewey D
authors.initials: DD
authors.lastname: Steadman
authors.forename: Sarah
authors.initials: S
authors.lastname: Durkin
authors.forename: Amy
authors.initials: A
authors.lastname: Baldwin
authors.forename: Wade H
authors.initials: WH
authors.lastname: Jeffrey
authors.forename: Peter
authors.initials: P
authors.lastname: Sheridan
authors.forename: Rene
authors.initials: R
authors.lastname: Horton
authors.forename: Malcolm S
authors.initials: MS
authors.lastname: Shields
mesh_terms: D000864:Antarctic Regions; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004586:Electrophoresis; D016983:Enterococcus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005431:Florida; D019015:Geologic Sediments; D016133:Polymerase Chain Reaction / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D012988:Soil Microbiology; D013698:Templates, Genetic
chemical_list: D004269:DNA, Bacterial