Molecular biology : a project approach

METHODS LOCATOR xiii SUGGESTED SCHEDULE OF LABORATORY PROTOCOLS xv PREFACE xvii ACKNOWLEDGMENTS xix NOTE TO USERS xxi 1 TRANSPOSON MUTAGENESIS OF Escherichia coli Introduction to Transposons 1 Advantages of Transposon Mutagenesis 6 Eukaryotic Transposable Elements 8 Transposons and Gene Fusions 9 Preparing for Laboratory Exercises 9 Common Laboratory Rules 9 Guidelines for Laboratory Notebooks 10 Guidelines for Laboratory Reports 1 1 Using Micropipettors 1 1 Review of Sterile Technique 12 Tn5 Mutagenesis of Escherichia coil and Analysis of Auxotrophs: Overview 15 Strain List 15 Media Recipes 17 Protocol 1.1: Phage ~ Titer 19 Protocol 1.2: Making a Phage Stock--Growing X-TnS' 21 Part A: Making Fresh Plaques 21 Part B: Making Phage Lysate Stocks 23 Protocol 1.3: Transposon Mutagenesis Using ~::TnphoA'-2 25 Introduction to Auxotrophs 27 Protocol 1.4: Isolation of Auxotrophs--Replica Plating, Toothpicking, or Screening on 2 EM Plates 27 Protocol 1.5: Identification of Auxotrophs on Pool Plates 31 Making Pool Plates 31 Protocol 1.6: Analysis of Auxotrophs Using a Literature Search 33 Genetic Mapping Strategies 34 References 35 Suggested Reading 36 2 RECOMBINANT DNA CLONING Introduction to Recombinant DNA Technology 45 Cloning Vectors 49 pBR322 52 Vectors That Yield Single-Stranded DNA 54 Development of the pUC Plasmids 56 Vectors for Cloning Large DNA Fragments 60 Restriction Endonucleases 63 Type I or Class I Restriction Endonucleases 65 Type II or Class II Restriction Endonucleases 66 Type III or Class III Restriction Endonucleases 70 Other Restriction Endonucleases 70 The Use of Restriction Endonucleases: Practical Matters Different Restriction Endonucleases 73 Setting Up a Restriction Digestion 73 Stopping a Restriction Digestion 76 Ligase 77 Gel Electrophoresis 78 71 Structure of Agarose 79 Pulsed Field Gel Electrophoresis 84 Capillary Electrophoresis 84 Ethidium Bromide Staining of DNA in Gels Ethidium Bromide Safety 86 Sensitivity of Detection with Ethidium Bromide Other DNA Stains 87 Transformation 88 Background 88 Transformation Procedures 90 Recombinant DNA Cloning: Overview 92 Description of pUC Vectors 93 /3-Galactosidase 93 85 87 The Insert DNA to Be Cloned: Origin and Significance of Cosmid 203 95 Alternative DNAs to Clone 96 Recombinant DNA: P1 Level of Physical Containment-- Laboratory Practices 97 Protocol 2. la (Optional): Restriction Digestion of DNA Samples and Gel Electrophoresis of DNA Samples 97 Protocol 2.1 b (Optional): Restriction Enzyme Digestion of DNA to Be Cloned 98 Protocol 2.2 (Optional): Gel Electrophoresis 99 Protocol 2.3: Large-Scale Plasmid Isolation Using Alkaline Lysis 102 Determination of DNA Concentration 109 Protocol 2.4: Recombinant DNA Cloning 110 Schedule for Recombinant DNA Cloning Experiment 110 Restriction Digestions for Cloning 111 Bacterial Transformation 114 Protocol 2.5a: Competent Escherichia coli Cells 116 Preparing and Freezing Competent Cells 116 Protocol 2.5b: Preparing Fresh Competent Escherichia coil Cells for Transformation 117 Using Competent Cells for Transformation 118 Protocol 2.5c: A Rapid Colony Transformation Procedure 119 Protocol 2.6a: Boiling Mini-Prep Isolation of Plasmid DNA 120 Protocol 2.6b: Alkaline Mini-Prep Procedure for Isolating Plasmid DNA 121 References 123 Suggested Reading 131 3 SOUTHERN BLOT ANALYSIS Southern Blot Introduction 135 Using Southern Blot Analysis to Map Restriction Endonuclease Sites 136 Nonradioactive Labeling of Nucleic Acids 136 Horseradish Peroxidase and Enhanced Chemiluminescence Digoxigenein Nonradioactive Labeling System 138 Biotin-Streptavidin Labeling System 139 Chromogenic Substrate for Alkaline Phosphatase 141 Chemiluminogenic Substrate for Alkaline Phosphatase 141 Autoradiography: Overview 143 Isolation of Nucleic Acid Fragments from Gels 145 Labeling Methods 147 Nick Translation 147 Oligo Labeling 150 Photobiotin 152 Hybridization to Membranes 153 Blot of a Dry Gel 155 The Attachment of Nucleic Acids to a Membrane 156 Protocol 3.1 a: Southern Blot 157 Modifications of Standard Blotting Procedures 160 Mini-Southern Blotting 160 Protocol 3.1 b: Bidirectional Blotting: A Sandwich Blot 161 Protocol 3.1 c: Alkaline Blotting 161 Protocol 3.1 d: Colony Hybridization 162 Protocol 3.2: Isolation of DNA Fragments by Electroelution Overview 164 Preparation of Dialysis Tubing 167 Labeling DNA to Be Used as Probes 169 Protocol 3.3a: Labeling Probe with Biotin Using Nick Translation 169 Separation of the Biotin-Labeled DNA from the Uncorporated Biotin-14-dATP by Exclusion Chromatography Using a Sephadex G-100 Column 170 Protocol 3.3b: Oligo Labeling of a Probe 171 Protocol 3.3c: Protobiotin Labeling of a Probe 173 Protocol 3.4a: Hybridization and Detection of Labeled Probe--A Biotin-Labeled Nonradioactive Probe and Chromogenic Substrate 174 137 164 Hybridization for a Chromogenic Nonradioactive Detection System 174 Detection of a Biotin-Labeled Probe for a Chromogenic Nonradioactive Detection System 176 Protocol 3.4b: Hybridization and Detection of Labeled Probe--A Biotin-Labeled Nonradioactive Probe and Chemiluminogenic Substrate 178 Additional Notes about Nonradioactive DNA Detection Systems 184 Protocol 3.5: Standard Southern Blot Hybridization with 32p-Labeled Probe 187 References 189 Suggested Reading 192 4 PLANT GENOMIC SOUTHERN BLOTTING WITH PROBES FOR LOW- AND HIGH-COPY-NUMBER GENES Overview of Experiment 193 Genomic Southern 193 Protocol 4.1: Plant DNA Extraction Mini-Prep Procedure 195 "Reconstructions" for Gels 196 Protocol 4.2: Steps of a Genomic Southern Blot 199 References 200 Suggested Reading 200 5 RNA PURIFICATION AND NORTHERN BLOT ANALYSIS RNA Introduction: Overview of Experiment 202 Protocol 5. I:RNA Extraction from Plant Leaves 203 Protocol 5.2: Separating Poly(A) § RNA from Total Cellular RNA 206 Batch Elution 206 Protocol 5.3: RNA Gel: A Denaturing Formaldehyde Gel 207 Protocol 5.4: A Northern Blot 208 Protocol 5.5: Standard Northern Blot Hybridization Conditions for 32p-Labeled Probe 210 References 213 Suggested Reading 213 6 POLYMERASE CHAIN REACTION Background 215 Protocol 6.1" PCR Experiment References 223 Suggested Reading 224 219 APPENDIX 1" Templates for Streaking Colonies 229 APPENDIX 2: Storing Bacterial Strains: Making Permanents 231 APPENDIX 3: Reporter Genes 235 APPENDIX 4: Antibiotic Information 239 APPENDIX 5: X-gal and IPTG 247 APPENDIX 6: More Information on Molecular Biology Protocols 249 APPENDIX 7: Sources of Strains 251 APPENDIX 8: List of Suppliers 253 APPENDIX 9: Additional Information 257 APPENDIX 10: Molecular Weight Standards 259 GLOSSARY 261 INDEX 275