Autoclave method for rapid preparation of bacterial PCR-template DNA.
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.
Simmon
Keith E
KE
The Center for Environmental Diagnostics and Bioremediation, The University of West Florida, Pensacola, FL, USA.
Steadman
Dewey D
DD
Durkin
Sarah
S
Baldwin
Amy
A
Jeffrey
Wade H
WH
Sheridan
Peter
P
Horton
Rene
R
Shields
Malcolm S
MS
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
D004269:DNA, Bacterial