Unraveling DNA : Molecular Biology for the Laboratory

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This innovative manual introduces students to all of the basic techniques of modern molecular biology using an integrated series of laboratory exercises that involve the cloning and analysis of the bioluminescence (lux) genes from the marine bacterium Vibrio fischeri. By organizing the exercises as part of a major cloning project, students get the sense of performing a complete cloning project, rather than just learning a collection of procedures. The manual is divided into discrete units with each demonstrating one or more aspects of the cloning project. Collectively, the entire series of exercises requires approximately three quarters to one full semester to complete with two laboratory periods per week. Smaller portions of the manuals are easily adapted to fewer lab periods.For Sale in Indian subcontinent onlyFeaturesConsists of an integrated series of exercises that culminate in the cloning and analysis of the genes coding for bacterial bioluminescence. The manual is based on one of nature's most fascinating biological phenomenon: the biological production of light. This results in a recurrent theme of interest for students and makes the project very relevant to interdisciplinary topics such as marine biology, ecology, biochemistry and biophysics. Includes instruction in the basic techniques of modern molecular biology: DNA isolation and analysis, DNA restriction, agarose gel electrophoresis, ligations, transformation of recombinant DNA, preparation and screening a genomic library, restriction mapping, Southern blotting, hybridization, DNA sequencing, pulsed field gel electrophoresis. All exercises have been thoroughly tested by hundreds of students and over 100 college faculty, including five years of NSF workshops. Includes extensive appendices on laboratory safety, the care and handling of enzymes, cloning vectors, and suppliers of equipment, materials and reagents.ContentsPart I: Introductory Techniques. Introduction to the Laboratory: Basic Equipment and Bacteriological Techniques. Preparation of Media and Reagents Used in Molecular Biology. Isolation of Luminescent Bacteria from Natural Sources. Restriction Digestion and Agarose Gel Electrophoresis of DNA. Part II: DNA Isolation and Analysis. Isolation of Chromosomal DNA from Vibrio fischeri Large-Scale Purification of Plasmid DNA. Spectrophotometric Analysis of DNA. Part III: Cloning the Lux Operon Restriction Digestion of Vibrio fischeri Genomic DNA and Plasmid Vector. Quantification of Genomic DNA by Fluorometry and Agarose Plate Fluorescence. Ligation of Restriction Fragments of Vibrio fischeri DNA to Plasmid Vector. Preparation of Competent Escherichia coli DH5. Transformation of Competent Escherichia coli DH5 with Recombinant Plasmids. Screening the Vibrio fischeri Genomic Library for Light Producing Clones. Part IV: Restriction Mapping and Southern Blotting Small-Scale Plasmid Isolations (Mini-preps) from Bioluminescent Clones. Restriction Mapping of Plasmids from Bioluminescent Clones. Southern Blotting and Hybridization to Detect the luxA Gene. Part V: Subcloning the Lux a Gene Restriction Digestion of lux Plasmids and Cloning Vector for Subcloning luxA. Gel Purification of DNA Restriction Fragments Containing luxA. Subcloning luxA into A Plasmid Vector. Transformation of Competent Escherichia coli DH5 with Subcloned DNA. Colony Hybridization to Screen for luxA Subclones. Small-Scale Plasmid Isolations (Mini-Preps) from luxA Clones. Part VI: Advanced Techniques. Amplification of luxA from Natural Isolates by the Polymerase Chain Reaction (PCR). Southern Blotting and Hybridization of PCR Products. DNA Sequencing of lux Genes from Plasmid Templates. Computer Analysis of DNA Sequences Using the World Wide Web. Mapping the Vibrio fischeri Genome by Pulsed Field Gel Electrophoresis. Independent Projects in Molecular Biology.