Basic Methods in Molecular Biology

ForewordAcknowledgments 1. The Basics of Molecular Biology 2. The Tools of the Molecular Biologist 3. General Preparations, Procedures, and Considerations for Use of Manual Section 3-1 Using This Manual 3-2 Safety Considerations 3-3 Equipment Needed for Molecular Biology Studies 4. Cloning Vectors and Bacterial Cells Section 4-1 pBR322 4-2 M13 4-3 pUC 4-4 λgtlO 4-5 λgtll 4-6 EMBL3 and EMBL4 4-7 Charon 28 4-8 Bacterial Strains5. Preparation of DNA from Eukaryotic Cells Section 5-1 Rapid DNA Preparation 5-2 Preparation of DNA from Eukaryotic Cells: General Method 5-3 DNA Preparation from Cultured Cells and Tissue 5-4 Restriction Endonucleases (REs) and Their Use 5-5 Agarose Gel Electrophoresis 5-6 Southern Blot 6. Probing Nucleic Acids with Labeled Synthetic Probes Section 6-1 Making Synthetic DNA Probes: General Description 6-2 End Labeling of Synthetic Probes 6-3 Hybridization with Synthetic 32P End-Labeled Probe7. Probing Nucleic Acids with Plasmid-Derived Probes Section 7-1 Nick Translation 7-2 DNA Hybridization (Southern Blot Hybridization)8. Plasmid DNA Preparation Section 8-1 Transformation of Bacteria 8-2 Plasmid DNA Preparation: Triton-Lysozyme Method 8-3 Large-Scale Alkaline Lysis Method: Plasmid Purification 8-4 Plasmid "Mini-Prep" Method 9. DNA Restriction Fragment Preparation Section 9-1 Minigels 1 9-2 Analysis of DNA Fragments After Enzymatic Cleavage: Agarose Gel Electrophoresis 9-3 Electroelution 9-4 Polyacrylamide Gel Electrophoresis of DNA Restriction Fragments 10. Purification of DNA Section 10-1 Spermine Purification of DNA 10-2 Glass Powder Elution of DNA 10-3 Purification of DNA: Other Methods 11. Preparation and Analysis of RNA from Eukaryotic Cells Section 11-1 Guanidine Isothiocyanate Preparation of Total RNA 11-2 RNA Preparation: Mini Method 11-3 Selection of Poly(A+) RNA on Oligo(dT) Cellulose 11-4 Formaldehyde Gel for Electrophoretic Separation of RNA and Northern Blot 11-5 "Dot Blot" Hybridization of Labeled Probe to DNA or RNA Samples 11-6 Probing RNA Gels: General Notes 11-7 Preparation of RNA Probes from DNA Cloned into Plasmids 12. Preparation of DNA from Bacteriophage Clones Section 12-1 Growth and Preparation of Bacteriophage 12-2 Large-Scale Preparation and Purification of DNA from Bacteriophage 13. Cloning DNA from the Eukaryotic Genome Section 13-1 Cloning DNA from the Eukaryotic Genome: Introduction 13-2 Preparation of Genomic DNA: Partial Mbol Digestion Method 13-3 Preparation of Bacteriophage Vector for Genomic Cloning 13-4 Ligation of Genomic DNA into Bacteriophage Arms and Packaging to Form Library 13-5 Titering and Plating of Packaged Library 13-6 Screening a Plated Library with Radiolabeled Probes 13-7 Library Amplification 14. cDNA Cloning into λgtlO and λgt11 Section 14-1 Preparation of λgt10 and λgt11 cDNA Cloning Vectors 14-2 Generation of cDNA Insert from Eukaryotic mRNA 14-3 Ligation and Packaging of cDNA Library into λgt10 or λgt11 Arms 14-4 Plating and Screening of λgt10 and λgt11 Packaged Inserts 14-5 Preparation of DNA from λgt10 and λgt11 cDNA Clones 15. Subcloning into Plasmids Section 15-1 Subcloning into Plasmids: General Notes 15-2 Preparing pBR322 Plasmids for Subcloning and Ligation of Insert 15-3 pBR322 Colony Hybridization 15-4 Subcloning into pUC Plasmids 16. M13 Cloning and Sequencing Section 16-1 M13 Cloning and Sequencing: General Notes 16-2 Preparation of Insert for Cloning from Specific Restriction Sites 16-3 Preparation of Insert for Μ13 Cloning by Successive BAL 31 Exonuclease Deletion 16-4 Μ13 Vector Preparation and Ligation of Insert into Vector 16-5 Transformation of M13 into JM103 E. coli Host 16-6 Screening Μ13 Clones with a Radiolabeled Probe to Select Inserts for Sequencing 16-7 Preparation of Single-Stranded Μ13 DNA for Sequencing 16-8 Single-Lane Screen Analysis of Μ13 Clones 16-9 Preparation of Polyacrylamide Sequencing Gel 16-10 Sequencing Μ13 Clones17. Further Characterization of Cloned DNA Section 17-1 Si Nuclease Protection Assay 18. Transfection of Mammalian Cells in Culture Section 18-1 Calcium Phosphate Transfection of Nonadherent and Adherent Cells with Purified Plasmids 18-2 DEAE Dextran-Mediated Transfection of Nonadherent and Adherent Mammalian Cells 18-3 Electroporation 18-4 Selection of Transfected Mammalian Cells: The G418 Method 18-5 Chloramphenicol Acetyltransferase (CAT) Assay 19. Protein Methods Section 19-1 In Vitro Translation and Immunoprecipitation 19-2 Polyacrylamide Gels for Protein Separation 19-3 Western Blot Analysis 19-4 Silver Staining of Gels for Proteins or RNA 20. General Methods Section 20-1 DNA/RNA Extraction and Precipitation 20-2 Plastic Bag Sealing 20-3 Optical Density Analytical Measurements 20-4 Photographing Gels or Autoradiograms 20-5 Autoradiography 20-6 Making Plates for Bacterial Growth 20-7 Titering and Plating of Phage 21. Specialized Methods Section 21-1 Transgenic Mouse Preparation 21-2 Monoclonal Antibody Production: Hybridoma Fusion 21-3 In Situ Hybridization of Labeled Probes to Tissue Sections 21-4 Cloning into Yeast Appendix I Stock Solutions Appendix II Enzymes Appendix III Suppliers of Reagents and Equipment Index