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This volume has evolved from a laboratory methods book that one of us first compiled nearly fifteen years ago. Since that time the book has undergone many minor revisions in order to include new methods and updated versions of older methods. The result has been an increasingly useful and more widely circulated book. However, the recent series of technological explosions generally lumped together under the name of "recombinant DNA technology" has been a turning point in the evolution of this previously underground publication. Minor revisions will no longer do. To keep the book useful we have…mehr
This volume has evolved from a laboratory methods book that one of us first compiled nearly fifteen years ago. Since that time the book has undergone many minor revisions in order to include new methods and updated versions of older methods. The result has been an increasingly useful and more widely circulated book. However, the recent series of technological explosions generally lumped together under the name of "recombinant DNA technology" has been a turning point in the evolution of this previously underground publication. Minor revisions will no longer do. To keep the book useful we have had to make major revi sions and additions. The result is a dramatically expanded book that should be more useful to more people. The larger size and wider usefulness of the book have made this more formal publication seem a reasonable step to take. One of the reasons that this volume should be useful to many people is that it includes only procedures that have been used repeatedly by us and that have proven highly reliable both to ourselves and to others in our laboratories.
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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.
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.
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