Molecular biology: A project approach

Front Cover......Page 1
Molecular Biology: A Project Approach......Page 4
Copyright Page......Page 5
CONTENTS......Page 8
METHODS LOCATOR......Page 14
SUGGESTED SCHEDULE OF LABORATORY PROTOCOLS......Page 16
PREFACE......Page 18
ACKNOWLEDGMENTS......Page 20
NOTE TO USERS......Page 22
Introduction to Transposons......Page 24
Advantages of Transposon Mutagenesis......Page 29
Eukaryotic Transposable Elements......Page 31
Preparing for Laboratory Exercises......Page 32
Tn5 Mutagenesis of Escherichia coil and Analysis of Auxotrophs: Overview......Page 38
Protocol 1.1: Phage λ Titer......Page 42
Protocol 1.2: Making a Phage Stock—Growing λ-Tn5'......Page 44
Protocol 1.3: Transposon Mutagenesis Using λ::TnphoA'-2......Page 48
Protocol 1.4: Isolation of Auxotrophs–Replica Plating, Toothpicking, or Screening on 2 EM Plates......Page 50
Protocol 1.5: Identification of Auxotrophs on Pool Plates......Page 54
Protocol 1.6: Analysis of Auxotrophs Using a Literature Search......Page 56
References......Page 58
Suggested Reading......Page 59
Introduction to Recombinant DNA Technology......Page 68
Cloning Vectors......Page 72
Restriction Endonucleases......Page 86
The Use of Restriction Endonucleases: Practical Matters......Page 94
Ligase......Page 100
Gel Electrophoresis......Page 101
Ethidium Bromide Staining of DNA in Gels......Page 108
Ethidium Bromide Safety......Page 109
Other DNA Stains......Page 110
Transformation......Page 111
Recombinant DNA Cloning: Overview......Page 115
Protocol 2. la (Optional): Restriction Digestion of DNA Samples and Gel Electrophoresis of DNA Samples......Page 120
Protocol 2.1 b (Optional): Restriction Enzyme Digestion of DNA to Be Cloned......Page 121
Protocol 2.2 (Optional): Gel Electrophoresis......Page 122
Protocol 2.3: Large-Scale Plasmid Isolation Using Alkaline Lysis......Page 125
Protocol 2.4: Recombinant DNA Cloning......Page 133
Protocol 2.5a: Competent Escherichia coli Cells......Page 139
Protocol 2.5b: Preparing Fresh Competent Escherichia coli Cells for Transformation......Page 140
Protocol 2.5c: A Rapid Colony Transformation Procedure......Page 142
Protocol 2.6a: Boiling Mini-Prep Isolation of Plasmid DNA......Page 143
Protocol 2.6b: Alkaline Mini-Prep Procedure for Isolating Plasmid DNA......Page 144
References......Page 146
Suggested Reading......Page 154
Southern Blot Introduction......Page 158
Nonradioactive Labeling of Nucleic Acids......Page 159
Autoradiography: Overview......Page 166
Isolation of Nucleic Acid Fragments from Gels......Page 168
Labeling Methods......Page 170
Hybridization to Membranes......Page 176
The Attachment of Nucleic Acids to a Membrane......Page 179
Protocol 3.1 a: Southern Blot......Page 180
Protocol 3.1 c: Alkaline Blotting......Page 184
Protocol 3.1 d: Colony Hybridization......Page 185
Protocol 3.2: Isolation of DNA Fragments by Electroelution......Page 187
Protocol 3.3a: Labeling Probe with Biotin Using Nick Translation......Page 192
Protocol 3.3b: Oligo Labeling of a Probe......Page 194
Protocol 3.3c: Protobiotin Labeling of a Probe......Page 196
Protocol 3.4a: Hybridization and Detection of Labeled Probe—A Biotin-Labeled Nonradioactive Probe and Chromogenic Substrate......Page 197
Protocol 3.4b: Hybridization and Detection of Labeled Probe—A Biotin-Labeled Nonradioactive Probe and Chemiluminogenic Substrate......Page 201
Protocol 3.5: Standard Southern Blot Hybridization with 32p-Labeled Probe......Page 210
References......Page 212
Suggested Reading......Page 215
Overview of Experiment......Page 216
Protocol 4.1: Plant DNA Extraction Mini-Prep Procedure......Page 218
"Reconstructions" for Gels......Page 219
Protocol 4.2: Steps of a Genomic Southern Blot......Page 222
Suggested Reading......Page 223
RNA Introduction: Overview of Experiment......Page 225
Protocol 5.1: RNA Extraction from Plant Leaves......Page 226
Protocol 5.2: Separating Poly(A) + RNA from Total Cellular RNA......Page 229
Protocol 5.3: RNA Gel: A Denaturing Formaldehyde Gel......Page 230
Protocol 5.4: A Northern Blot......Page 231
Protocol 5.5: Standard Northern Blot Hybridization Conditions for 32p-Labeled Probe......Page 233
Suggested Reading......Page 236
Background......Page 238
Protocol 6.1: PCR Experiment......Page 242
References......Page 246
Suggested Reading......Page 247
APPENDIX 1: Templates for Streaking Colonies......Page 252
APPENDIX 2: Storing Bacterial Strains: Making Permanents......Page 254
APPENDIX 3: Reporter Genes......Page 258
APPENDIX 4: Antibiotic Information......Page 262
APPENDIX 5: X-gal and IPTG......Page 270
APPENDIX 6: More Information on Molecular Biology Protocols......Page 272
APPENDIX 7: Sources of Strains......Page 274
APPENDIX 8: List of Suppliers......Page 276
APPENDIX 9: Additional Information......Page 280
APPENDIX 10: Molecular Weight Standards......Page 282
GLOSSARY......Page 284
INDEX......Page 298