Laboratory Exercises and Appendices

Unraveling DNA: Molecular Biology for the Laboratory

Michael R. Winfrey, Marc A. Rott, and Alan T. Wortman.

I. Introductory Techniques

Ex 1. Introduction to the Laboratory: Basic Equipment and Bacteriological Techniques

Ex 2. Preparation of Media and Reagents Used in Molecular Biology

Ex 3. Isolation of Luminescent Bacteria from Natural Sources

Ex 4. Restriction Digestion and Agarose Gel Electrophoresis of DNA

II. DNA Isolation and Analysis

Ex 5. Isolation of Chromosomal DNA from Vibrio fischeri

Ex 6. Large Scale Purification of Plasmid DNA

Ex 7. Spectrophotometric Analysis of DNA

III. Cloning the lux operon

Ex 8. Restriction Digestion of Vibrio fischeri Genomic DNA and Plasmid Vector

Ex 9. Quantification of Genomic DNA by Fluorometry and Agarose Plate Fluorescence

Ex 10. Ligation of Restriction Fragments of Vibrio fischeri DNA to a Plasmid Vector

Ex 11. Preparation of Competent Escherichia coli DH5a

Ex 12. Transformation of Competent Escherichia coli DH5a with Recombinant Plasmids

Ex 13. Screening Vibrio fischeri Genomic Library for Light Producing Clones

IV. Restriction Mapping and Southern Blotting

Ex 14. Small Scale Plasmid Isolations (Mini-preps) from Bioluminescent Clones

Ex 15. Restriction Mapping of Plasmids from Bioluminescent Clones

Ex 16. Southern Blotting and Hybridization to Detect the luxA Gene

V. Subcloning the luxA gene

Ex 17. Restriction Digestion of lux Plasmids and Cloning Vector for Subcloning luxA

Ex 18. Gel Purification of DNA Restriction Fragments Containing luxA

Ex 19. Subcloning luxA into a Plasmid Vector

Ex 20. Transformation of Competent Escherichia coli DH5a with Subcloned DNA

Ex 21. Colony Hybridization to Screen for luxA Subclones

Ex 22. Small Scale Plasmid Isolations (Mini-preps) from luxA Clones

VI. Advanced Techniques

Ex 23. Amplification of luxA from Natural Isolates by the Polymerase Chain Reaction (PCR)

Ex 24. Southern Blotting and Hybridization of PCR Products

Ex 25. DNA Sequencing of lux Genes from Plasmid Templates

Ex 26. Computer Analysis of DNA Sequences Using the World Wide Web

Ex 27. Mapping the Vibrio fischeri Genome by Pulsed Field Gel Electrophoresis

Ex 28. Independent Projects in Molecular Biology

Appendices

I.The Metric System and Units of Measure
II. Centrifugation
III. Spectrophotometry
IV. Agarose and Polyacrylamide Gel Electrophoresis
V. Methylene Blue Staining of Agarose Gels
VI. Nucleic Acid Hybridization
VII. Alcohol Precipitation of Nucleic Acids
VIII. Care and Handling of Enzymes
IX. Restriction Endonucleases
X. Enzymes Used in Molecular Biology
XI. Maps of Cloning Vectors and Bacteriophage Lambda
XII. Proper Handling and Disposal of Hazardous Materials
XIII. Use of Radioisotopes
XIV. Equipment and Supplies
XV. Media and Reagents
XVI. Bacterial Strains
XVII. Lists of Suppliers
XVIII. Recommended References
XIX. Restriction Mapping Problems

Please address your questions,comments and suggestions concerning the manual to Mike Winfrey or Marc Rott

Created by Tom Volk

Last Updated: March 22, 1997