Unraveling DNA

<p> <strong>I. INTRODUCTORY TECHNIQUES.</strong></p> <p> <strong> 1. Introduction to the Laboratory: Basic Equipment and Bacteriological Techniques.</strong></p> <p> <br></p> <p> <strong> 2. Preparation of Media and Reagents Used in Molecular Biology.</strong></p> <p> <br></p> <p> <strong> 3. Isolation of Luminescent Bacteria from Natural Sources.</strong></p> <p> <br></p> <p> <strong> 4. Restriction Digestion and Agarose Gel Electrophoresis of DNA.</strong></p> <p> <br></p> <p> <strong>II. DNA ISOLATION AND ANALYSIS.</strong></p> <p> <strong> 5. Isolation of Chromosomal DNA from Vibrio fischeri. </strong></p> <p> <br></p> <p> <strong> 6. Large-Scale Purification of Plasmid DNA.</strong></p> <p> <br></p> <p> <strong> 7. Spectrophotometric Analysis of DNA.</strong></p> <p> <br></p> <p> <strong>III. CLONING THE LUX OPERON</strong></p> <p> <strong> 8. Restriction Digestion of Vibrio fischeri Genomic DNA and Plasmid Vector.</strong></p> <p> <br></p> <p> <strong> 9. Quantification of Genomic DNA by Fluorometry and Agarose Plate Fluorescence.</strong></p> <p> <br></p> <p> <strong>10. Ligation of Restriction Fragments of Vibrio fischeri DNA to Plasmid Vector.</strong></p> <p> <br></p> <p> <strong>11. Preparation of Competent Escherichia coli DH5.</strong></p> <p> <br></p> <p> <strong>12. Transformation of Competent Escherichia coli DH5 with Recombinant Plasmids.</strong></p> <p> <br></p> <p> <strong>13. Screening the Vibrio fischeri Genomic Library for Light Producing Clones.</strong></p> <p> <br></p> <p> <strong>IV. RESTRICTION MAPPING AND SOUTHERN BLOTTING</strong></p> <p> <strong>14. Small-Scale Plasmid Isolations (Mini-preps) from Bioluminescent Clones.</strong></p> <p> <br></p> <p> <strong>15. Restriction Mapping of Plasmids from Bioluminescent Clones.</strong></p> <p> <br></p> <p> <strong>16. Southern Blotting and Hybridization to Detect the luxA Gene.</strong></p> <p> <br></p> <p> <strong>V. SUBCLONING THE LUX A GENE</strong></p> <p> <strong>17. Restriction Digestion of lux Plasmids and Cloning Vector for Subcloning luxA.</strong></p> <p> <br></p> <p> <strong>18. Gel Purification of DNA Restriction Fragments Containing luxA.</strong></p> <p> <br></p> <p> <strong>19. Subcloning luxA into A Plasmid Vector.</strong></p> <p> <br></p> <p> <strong>20. Transformation of Competent Escherichia coli DH5 with Subcloned DNA.</strong></p> <p> <br></p> <p> <strong>21. Colony Hybridization to Screen for luxA Subclones.</strong></p> <p> <br></p> <p> <strong>22. Small-Scale Plasmid Isolations (Mini-Preps) from luxA Clones.</strong></p> <p> <br></p> <p> <strong>VI. ADVANCED TECHNIQUES.</strong></p> <p> <strong>23. Amplification of luxA from Natural Isolates by the Polymerase Chain Reaction (PCR).</strong></p> <p> <br></p> <p> <strong>24. Southern Blotting and Hybridization of PCR Products.</strong></p> <p> <br></p> <p> <strong>25. DNA Sequencing of lux Genes from Plasmid Templates.</strong></p> <p> <br></p> <p> <strong>26. Computer Analysis of DNA Sequences Using the World Wide Web.</strong></p> <p> <br></p> <p> <strong>27. Mapping the Vibrio fischeri Genome by Pulsed Field Gel Electrophoresis.</strong></p> <p> <br></p> <p> <strong>28. Independent Projects in Molecular Biology.</strong></p> <p> <br></p> <p> <strong>APPENDICES.</strong></p> <p> <strong> 1. The Metric System and Units of Measure.</strong></p> <p> <br></p> <p> <strong> 2. Centrifugation.</strong></p> <p> <br></p> <p> <strong> 3. Spectrophotometry.</strong></p> <p> <br></p> <p> <strong> 4. Agarose and Polyacrylamide Gel Electrophoresis.</strong></p> <p> <br></p> <p> <strong> 5. Methylene Blue Staining of Agarose Gels.</strong></p> <p> <br></p> <p> <strong> 6. Nucleic Acid Hybridization.</strong></p> <p> <br></p> <p> <strong> 7. Alcohol Precipitation of Nucleic Acids.</strong></p> <p> <br></p> <p> <strong> 8. Care and Handling of Enzymes.</strong></p> <p> <br></p> <p> <strong> 9. Restriction Endonucleases.</strong></p> <p> <br></p> <p> <strong>10. Enzymes Used in Molecular Biology.</strong></p> <p> <br></p> <p> <strong>11. Maps of Cloning Vectors and Bacteriophage Lambda.</strong></p> <p> <br></p> <p> <strong>12. Proper Handling and Disposal of Hazardous Materials.</strong></p> <p> <br></p> <p> <strong>13. Procedures and Precautions for the Use of Radioisotopes.</strong></p> <p> <br></p> <p> <strong>14. Equipment and Supplies.</strong></p> <p> <br></p> <p> <strong>15. Media and Reagents.</strong></p> <p> <br></p> <p> <strong>16. Bacterial Strains.</strong></p> <p> <br></p> <p> <strong>17. Lists of Suppliers.</strong></p> <p> <br></p> <p> <strong>18. Recommended References.</strong></p> <p> <br></p> <p> <strong>19. Restriction Mapping Problems.</strong></p>