Practical Methods in Molecular Biology

1 Using E. coli.- Strains.- Strain Purity.- Sources of Strains.- Commonly Used Strains.- Pedigrees and Genetic Maps.- Strains for Physiological Measurements.- Storage of Strains.- Cell Growth.- Properties of Bacteria.- Phage Contamination.- Use of Cells for Physiological Measurements.- Use of Cells for Genetic Purposes.- Growing Cells for the Purification of Molecules.- Measuring Cell Density.- Growing Large Quantities of Cells.- Opening Cells.- Sonicating.- Grinding in Alumina.- Grinding with Glass Beads.- Opening Cells with Lysozyme.- Radiolabelling Cells.- Nitrosoguanidine Mutagenesis.- Penicillin Selection.- Curing Cells of F-Factors.- Curing with Acridine Orange.- Selecting Spontaneously Cured Cells.- Curing with Sodium Dodecyl Sulfate.- Curing with High Temperature.- Making Cells Streptomycin-Resistant.- Crossing recA into Cells.- Phage P1 Transduction of Genetic Markers.- Large-Scale Genetic Crosses.- Using Transposons in Strain Construction.- 2 Bacteriophage Lambda.- Two Useful Mutants.- Cl857.- S7.- Titering.- Growing Plate Stocks.- Large-Scale Growth in Liquid.- Purification.- Genetic Crosses.- Scoring Plaques.- Making Strains Lambda-Resistant.- Testing Colonies for the Ability to Grow Lambda.- Making Lysogens.- Testing Lysogen Candidates.- Streaking for Single Plaques.- Selecting Deletions.- 3 Enzyme Assays.- ?-Galactosidase.- RNA Polymerase.- Arabinose Isomerase.- Lysozyme.- Ribulokinase.- E. coli-Coupled Transcription-Translation System.- 4 Working with Proteins.- Ammonium Sulfate Precipitation of Proteins.- Removing Nucleic Acids by Phase Partition.- Bulk Separation of Proteins from Nucleic Acids.- Purification of Protein-Bound DNA or RNA.- Columns, Fraction Collectors, and Plumbing.- Ion Exchange Chromatography and Gel Filtration.- Preparing Ion Exchange Resins.- Pouring Columns.- Loading and Eluting Columns.- Determining Protein Concentration.- Optical Density.- Biuret.- Lowry.- Lowry for Dilute Samples.- Concentrating Protein Solutions.- Stabilizing Proteins.- Polyacrylamide Gel Electrophoresis of Proteins.- Making a Gel Sandwich.- SDS-10% Acrylamide Gel with Stacking Gel.- Urea-SDS-Acrylamide Gradient Gels.- Staining Gels.- Fluorography of [3H]- or [35S]-Labelled Proteins in Acrylamide Gels.- Recovering PPO from Solution in DMSO.- 5 Working with Nucleic Acids.- Measuring Nucleic Acid Concentration and Purity.- Optical Methods.- Fluorescence Method.- Storing DNA.- Cleaning DNA.- Cleaning by DEAE.- Cleaning by Hydroxyapatite.- Precipitating DNA with Ethanol.- TCA Precipitation Assay.- Precipitation and Size Fractionation of DNA with Poly-ethylene Glycol.- Isolating E. coli DNA.- Isolating Lambda DNA.- Phenol Extraction.- SDS Extraction.- Isolating Plasmid DNA.- Large-Scale Plasmid Isolation.- Isolating Drosophila DNA.- Preparing Nucleoside Triphosphate Solutions.- Chromatographic Analysis of Nucleosides.- Gel Electrophoresis of DNA.- Agarose Gel Electrophoresis.- Polyacrylamide Gel Electrophoresis.- Staining and Photographing Gels.- Extracting DNA from Acrylamide and Agarose Gels.- Mapping Restriction Endonuclease Sites on DNA.- 6 Constructing and Analyzing Recombinant DNA.- Joining the Ends of DNA Molecules.- Making Blunt Ends by S1 Digestion.- Ligation with T4 DNA Ligase.- Lambda Exonuclease Digestion to Yield Free 3' Ends.- Addition of Homopolymer Tails.- E. coli Transformation with Plasmid DNA.- Storing Strains that Contain Plasmids.- Cycloserine Selection of Recombinant Plasmids.- In Vitro Radiolabelling of DNA and RNA.- Radiolabelling DNA by Nick Translation.- Synthesizing Radiolabelled RNA Complementary to DNA.- Synthesizing Radiolabelled DNA Complementary to RNA.- Radiolabelling the 5' Ends of RNA or DNA.- General Aspects of Nucleic Acid Hybridization Reactions.- Screening Recombinant DNA Clones by Nucleic Acid Hybridization.- Screening Phage Plaques.- Screening Colonies.- Isolating DNA from a Single Colony.- Southern Transfers.- Selecting RNA Complementary to a DNA.- Preparing DNA.- Preparing NBM Paper.- Converting NBM Paper to DBM Paper and Binding the DNA.- Hybridizing RNA to DBM-Bound DNA.- Recovering the RNA.- Synthesis of NBPC (nitrobenzylpyridinium chloride).- In Vitro Translation Systems from Higher Organisms.- Preparing a Wheat Germ Extract.- Wheat Germ Translation Reaction.- Preparing a Rabbit Reticulocyte Lysate.- Micrococcal Nuclease Digestion of Lysate.- Reticulocyte Translation Reaction.- Purifying Total and Polysomal RNA.- Extracting RNA from Drosophila Adults.- Extracting Polysomal RNA from Drosophila Embryos.- Purification of Poly-A+ (mRNA-Enriched) RNA.- RNA Size Fractionation by Sucrose Gradient Centrifugation.- Making the Sucrose Gradients.- Preparing the RNA.- Sedimenting and Collecting the RNA.- 7 Assorted Laboratory Techniques.- Glass and Plastic Containers.- Siliconizing Glassware.- Washing Pipettes.- pH Meters.- Buffers (Tris, Phosphate, Good Buffers, Cacodylate).- Beckman Ultracentrifuges.- Filling and Balancing Tubes.- Loading Rotors.- Drawing Figures.- Slides and Negatives.- Polaroid Slides.- Conventional Slides and Negatives.- Film Sensitometry.- Autoradiography and Fluorography.- Dialysis Tubing.- Distilling Phenol.- Recovering Used CsCl.- Sources of Chemicals.- Hazards and Cautions.- Desiccators, Preparative Centrifuges, and Vacuum Pumps.- Chemicals.- Electrical Hazards and Protections.- Appendix I Commonly Used Recipes.- Appendix II Useful Numbers.