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| Methods Locator | |
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| Suggested Schedule of Laboratory Protocols | |
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| Preface | |
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| Acknowledgments | |
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| Note to Users | |
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| Transposon Mutagenesis of Escherichia coli | |
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| Introduction to Transposons | |
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| Advantages of Transposon Mutagenesis | |
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| Eukaryotic Transposable Elements | |
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| Transposons and Gene Fusions | |
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| Preparing for Laboratory Exercises | |
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| Common Laboratory Rules | |
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| Guidelines for Laboratory Notebooks | |
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| Guidelines for Laboratory Reports | |
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| Using Micropipettors | |
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| Review of Sterile Technique | |
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| Tn5 Mutagenesis of Escherichia coli and Analysis of Auxotrophs: Overview | |
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| Strain List | |
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| Media Recipes | |
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| Phage [lambda] Titer | |
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| Making a Phage Stock--Growing [lambda]-Tn5' | |
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| Making Fresh Plaques | |
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| Making Phage Lysate Stocks | |
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| Transposon Mutagenesis Using [lambda]:: TnphoA'-2 | |
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| Introduction to Auxotrophs | |
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| Isolation of Auxotrophs--Replica Plating, Toothpicking, or Screening on 2 EM Plates | |
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| Identification of Auxotrophs on Pool Plates | |
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| Making Pool Plates | |
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| Analysis of Auxotrophs Using a Literature Search | |
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| Genetic Mapping Strategies | |
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| References | |
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| Suggested Reading | |
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| Recombinant Dan Cloning | |
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| Introduction to Recombinant DNA Technology | |
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| Cloning Vectors | |
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| pBR322 | |
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| Vectors That Yield Single-Stranded DNA | |
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| Development of the pUC Plasmids | |
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| Vectors for Cloning Large DNA Fragments | |
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| Restriction Endonucleases | |
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| Type I or Class I Restriction Endonucleases | |
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| Type II or Class II Restriction Endonucleases | |
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| Type III or Class III Restriction Endonucleases | |
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| Other Restriction Endonucleases | |
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| The Use of Restriction Endonucleases: Practical Matters | |
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| Different Restriction Endonucleases | |
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| Setting Up a Restriction Digestion | |
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| Stopping a Restriction Digestion | |
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| Ligase | |
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| Gel Electrophoresis | |
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| Structure of Agarose | |
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| Pulsed Field Gel Electrophoresis | |
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| Capillary Electrophoresis | |
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| Ethidium Bromide Staining of DNA in Gels | |
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| Ethidium Bromide Safety | |
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| Sensitivity of Detection with Ethidium Bromide | |
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| Other DNA Stains | |
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| Transformation | |
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| Background | |
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| Transformation Procedures | |
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| Recombinant DNA Cloning: Overview | |
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| Description of pUC Vectors | |
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| [beta]-Galactosidase | |
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| The Insert DNA to Be Cloned: Origin and Significance of Cosmid 203 | |
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| Alternative DNAs to Clone | |
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| Recombinant DNA: P1 Level of Physical Containment--Laboratory Practices | |
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| Restriction Digestion of DNA Samples and Gel Electrophoresis of DNA Samples | |
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| Restriction Enzyme Digestion of DNA to Be Cloned | |
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| Gel Electrophoresis | |
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| Large-Scale Plasmid Isolation Using Alkaline Lysis | |
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| Determination of DNA Concentration | |
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| Recombinant DNA Cloning | |
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| Schedule for Recombinant DNA Cloning Experiment | |
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| Restriction Digestions for Cloning | |
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| Bacterial Transformation | |
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| Competent Escherichia coli Cells | |
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| Preparing and Freezing Competent Cells | |
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| Preparing Fresh Competent Escherichia coli Cells for Transformation | |
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| Using Competent Cells for Transformation | |
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| A Rapid Colony Transformation Procedure | |
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| Boiling Mini-Prep Isolation of Plasmid DNA | |
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| Alkaline Mini-Prep Procedure for Isolating Plasmid DNA | |
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| References | |
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| Suggested Reading | |
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| Southern Blot Analysis | |
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| Southern Blot Introduction | |
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| Using Southern Blot Analysis to Map Restriction Endonuclease Sites | |
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| Nonradioactive Labeling of Nucleic Acids | |
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| Horseradish Peroxidase and Enhanced Chemiluminescence | |
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| Digoxigenein Nonradioactive Labeling System | |
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| Biotin-Streptavidin Labeling System | |
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| Chromogenic Substrate for Alkaline Phosphatase | |
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| Chemiluminogenic Substrate for Alkaline Phosphatase | |
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| Autoradiography: Overview | |
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| Isolation of Nucleic Acid Fragments from Gels | |
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| Labeling Methods | |
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| Nick Translation | |
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| Oligo Labeling | |
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| Photobiotin | |
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| Hybridization to Membranes | |
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| Blot of a Dry Gel | |
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| The Attachment of Nucleic Acids to a Membrane | |
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| Southern Blot | |
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| Modifications of Standard Blotting Procedures | |
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| Mini-Southern Blotting | |
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| Bidirectional Blotting: A Sandwich Blot | |
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| Alkaline Blotting | |
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| Colony Hybridization | |
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| Isolation of DNA Fragments by Electroelution | |
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| Overview | |
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| Preparation of Dialysis Tubing | |
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| Labeling DNA to Be Used as Probes | |
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| Labeling Probe with Biotin Using Nick Translation | |
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| Separation of the Biotin-Labeled DNA from the Uncorporated Biotin-14-dATP by Exclusion Chromatography Using a Sephadex G-100 Column | |
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| Oligo Labeling of a Probe | |
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| Protobiotin Labeling of a Probe | |
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| Hybridization and Detection of Labeled Probe--A | |
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| Biotin-Labeled Nonradioactive Probe and Chromogenic Substrate | |
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| Hybridization for a Chromogenic Nonradioactive Detection System | |
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| Detection of a Biotin-Labeled Probe for a Chromogenic Nonradioactive Detection System | |
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| Hybridization and Detection of Labeled Probe--A Biotin-Labeled Nonradioactive Probe and Chemiluminogenic Substrate | |
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| Additional Notes about Nonradioactive DNA Detection Systems | |
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| Standard Southern Blot Hybridization with [superscript 32]P-Labeled Probe | |
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| References | |
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| Suggested Reading | |
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| Plant Genomic Southern Blotting With Probes for Low- and High-Copy-Number Genes | |
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| Overview of Experiment | |
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| Genomic Southern | |
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| Plant DNA Extraction Mini-Prep Procedure | |
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| "Reconstructions" for Gels | |
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| Steps of a Genomic Southern Blot | |
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| References | |
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| Suggested Reading | |
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| Rna Purification and Northern Blot Analysis | |
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| RNA Introduction: Overview of Experiment | |
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| RNA Extraction from Plant Leaves | |
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| Separating Poly(A)[superscript +] RNA from Total Cellular RNA | |
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| Batch Elution | |
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| RNA Gel: A Denaturing Formaldehyde Gel | |
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| A Northern Blot | |
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| Standard Northern Blot Hybridization Conditions for [superscript 32]P-Labeled Probe | |
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| Nonradioactive Biotin-Labeled Probes for Northern Blots | |
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| References | |
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| Suggested Reading | |
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| Polymerase Chain Reaction | |
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| Background | |
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| PCR Experiment | |
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| References | |
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| Suggested Reading | |
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| Templates for Streaking Colonies | |
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| Storing Bacterial Strains: Making Permanents | |
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| Reporter Genes | |
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| Antibiotic Information | |
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| X-gal and IPTG | |
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| More Information on Molecular Biology Protocols | |
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| Sources of Strains | |
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| List of Suppliers | |
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| Additional Information | |
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| Molecular Weight Standards | |
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| Glossary | |
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| Index | |