Basic Methods in Molecular Biology

Content: 
Front Matter, Page iii
Copyright, Page iv
Foreword, Page ix
Acknowledgments, Page xi
Introduction to The Basics of Molecular Biology, Pages 2-5
Introduction to The Tools of the Molecular Biologist, Pages 8-12
SECTION 3-1 - Using This Manual, Pages 14-16
SECTION 3-2 - Safety Considerations, Pages 17-18
SECTION 3-3 - Equipment Needed for Molecular Biology Studies, Pages 19-21
SECTION 4-1 - pBR322, Pages 24-25
SECTION 4-2 - M13, Pages 26-29
SECTION 4-3 - pUC, Pages 30-31
SECTION 4-4 - λgt10, Pages 32-33
SECTION 4-5 - λgt11, Pages 34-35
SECTION 4-6 - EMBL3 and EMBL4, Pages 36-37
SECTION 4-7 - Charon 28, Pages 38-39
SECTION 4-8 - Bacterial Strains, Page 40
SECTION 5-1 - Rapid DNA Preparation, Pages 42-43
SECTION 5-2 - Preparation of DNA from Eukaryotic Cells: General Method, Pages 44-46
SECTION 5-3 - DNA Preparation from Cultured Cells and Tissue, Pages 47-50
SECTION 5-4 - Restriction Endonucleases (REs) and Their Use, Pages 51-57
SECTION 5-5 - Agarose Gel Electrophoresis, Pages 58-61
SECTION 5-6 - Southern Blot, Pages 62-65
SECTION 6-1 - Making Synthetic DNA Probes: General Description, Pages 68-71
SECTION 6-2 - End Labeling of Synthetic Probes, Pages 72-74
SECTION 6-3 - Hybridization with Synthetic 32P End-Labeled Probe, Pages 75-78
SECTION 7-1 - Nick Translation, Pages 80-83
SECTION 7-2 - DNA Hybridization (Southern Blot Hybridization), Pages 84-87
SECTION 8-1 - Transformation of Bacteria, Pages 90-92
SECTION 8-2 - Plasmid DNA Preparation: Triton-Lysozyme Method, Pages 93-98
SECTION 8-3 - Large-Scale Alkaline Lysis Method: Plasmid Purification, Pages 99-101
SECTION 8-4 - Plasmid “Mini-Prep” Method, Pages 102-104
SECTION 9-1 - Minigels, Pages 106-108
SECTION 9-2 - Analysis of DNA Fragments After Enzymatic Cleavage: Agarose Gel Electrophoresis, Pages 109-111
SECTION 9-3 - Electroelution, Pages 112-114
SECTION 9-4 - Polyacrylamide Gel Electrophoresis of DNA Restriction Fragments, Pages 115-118
SECTION 10-1 - Spermine Purification of DNA, Pages 120-122
SECTION 10-2 - Glass Powder Elution of DNA, Pages 123-125
SECTION 10-3 - Purification of DNA: Other Methods, Pages 126-128
SECTION 11-1 - Guanidine Isothiocyanate Preparation of Total RNA, Pages 130-135
SECTION 11-2 - RNA Preparation: Mini Method, Pages 136-138
SECTION 11-3 - Selection of Poly(A+) RNA on Oligo(dT) Cellulose, Pages 139-142
SECTION 11-4 - Formaldehyde Gel for Electrophoretic Separation of RNA and Northern Blot, Pages 143-146
SECTION 11-5 - “Dot Blot” Hybridization of Labeled Probe to DNA or RNA Samples, Pages 147-149
SECTION 11-6 - Probing RNA Gels: General Notes, Pages 150-151
SECTION 11-7 - Preparation of RNA Probes from DNA Cloned into Plasmids, Pages 152-156
SECTION 12-1 - Growth and Preparation of Bacteriophage, Pages 158-160
SECTION 12-2 - Large-Scale Preparation and Purification of DNA from Bacteriophage, Pages 161-165
SECTION 13-1 - Cloning DNA from the Eukaryotic Genome: Introduction, Pages 168-170
SECTION 13-2 - Preparation of Genomic DNA: Partial Mbol Digestion Method, Pages 171-174
SECTION 13-3 - Preparation of Bacteriophage Vector for Genomic Cloning, Pages 175-179
SECTION 13-4 - Ligation of Genomic DNA into Bacteriophage Arms and Packaging to Form Library, Pages 180-181
SECTION 13-5 - Titering and Plating of Packaged Library, Pages 182-184
SECTION 13-6 - Screening a Plated Library with Radiolabeled Probes, Pages 185-189
SECTION 13-7 - Library Amplification, Pages 190-191
SECTION 14-1 - Preparation of λgt10 and λgt11 cDNA Cloning Vectors, Pages 194-198
SECTION 14-2 - Generation of cDNA Insert from Eukaryotic mRNA, Pages 199-207
SECTION 14-3 - Ligation and Packaging of cDNA Library into λgt10 or λgt11 Arms, Pages 208-210
SECTION 14-4 - Plating and Screening of λgt10 and λgt11 Packaged Inserts, Pages 211-215
SECTION 14-5 - Preparation of DNA from λgt10 and λgt11 cDNA Clones, Pages 216-218
SECTION 15-1 - Subcloning into Plasmids: General Notes, Pages 220-221
SECTION 15-2 - Preparing pBR322 Plasmids for Subcloning and Ligation of Insert, Pages 222-226
SECTION 15-3 - pBR322 Colony Hybridization, Pages 227-229
SECTION 15-4 - Subcloning into pUC Plasmids, Pages 230-232
SECTION 16-1 - M13 Cloning and Sequencing: General Notes, Pages 234-239
SECTION 16-2 - Preparation of Insert for Cloning from Specific Restriction Sites, Pages 240-243
SECTION 16-3 - Preparation of Insert for M13 Cloning by Successive BAL 31 Exonuclease Deletion, Pages 244-248
SECTION 16-4 - M13 Vector Preparation and Ligation of Insert into Vector, Pages 249-252
SECTION 16-5 - Transformation of M13 into JM103 E. coli Host, Pages 253-255
SECTION 16-6 - Screening M13 Clones with a Radiolabeled Probe to Select Inserts for Sequencing, Pages 256-257
SECTION 16-7 - Preparation of Single-Stranded M13 DNA for Sequencing, Pages 258-260
SECTION 16-8 - Single-Lane Screen Analysis of M13 Clones, Pages 261-263
SECTION 16-9 - Preparation of Polyacrylamide Sequencing Gel, Pages 264-267
SECTION 16-10 - Sequencing M13 Clones, Pages 268-273
SECTION 17-1 - S1 Nuclease Protection Assay, Pages 276-284
SECTION 18-1 - Calcium Phosphate Transfection of Nonadherent and Adherent Cells with Purified Plasmids, Pages 286-289
SECTION 18-2 - DEAE Dextran—Mediated Transfection of Nonadherent and Adherent Mammalian Cells, Pages 290-292
SECTION 18-3 - Electroporation, Pages 293-295
SECTION 18-4 - Selection of Transfected Mammalian Cells: The G418 Method, Pages 296-297
SECTION 18-5 - Chloramphenicol Acetyltransferase (CAT) Assay, Pages 298-300
SECTION 19-1 - In Vitro Translation and Immunoprecipitation, Pages 302-305
SECTION 19-2 - Polyacrylamide Gels for Protein Separation, Pages 306-310
SECTION 19-3 - Western Blot Analysis, Pages 311-314
SECTION 19-4 - Silver Staining of Gels for Proteins or RNA, Pages 315-317
SECTION 20-1 - DNA/RNA Extraction and Precipitation, Pages 320-323
SECTION 20-2 - Plastic Bag Sealing, Pages 324-326
SECTION 20-3 - Optical Density Analytical Measurements, Pages 327-328
SECTION 20-4 - Photographing Gels or Autoradiograms, Pages 329-330
SECTION 20-5 - Autoradiography, Pages 331-332
SECTION 20-6 - Making Plates for Bacterial Growth, Pages 333-335
SECTION 20-7 - Titering and Plating of Phage, Pages 336-337
SECTION 21-1 - Transgenic Mouse Preparation, Pages 340-347
SECTION 21-2 - Monoclonal Antibody Production: Hybridoma Fusion, Pages 348-354
SECTION 21-3 - In Situ Hybridization of Labeled Probes to Tissue Sections, Pages 355-359
SECTION 21-4 - Cloning into Yeast, Pages 360-362
APPENDIX I - Stock Solutions, Pages 363-369
APPENDIX II - Enzymes, Pages 370-371
APPENDIX III - Suppliers of Reagents and Equipment, Pages 372-376
Index, Pages 377-388