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Basic Methods in Molecular Biology
- 1st Edition - November 15, 2012
- Author: Leonard Davis
- Language: English
- Paperback ISBN:9 7 8 - 0 - 4 4 4 - 5 6 9 4 6 - 2
- eBook ISBN:9 7 8 - 0 - 4 4 4 - 6 0 1 4 9 - 0
Basic Methods in Molecular Biology discusses the heart of the most recent revolution in biology—the development of the technology of genetics. The achievements in this field have… Read more
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Request a sales quoteBasic Methods in Molecular Biology discusses the heart of the most recent revolution in biology—the development of the technology of genetics. The achievements in this field have simply changed what biologists do and, perhaps even more important, the way they think. Moreover, never before have scientists from such a broad range of disciplines rushed into such a small and slightly arcane field to learn and carry off a bit of the technology. This book comprises 21 chapters, opening with three introductory ones that discuss the basics of molecular biology; the tools of the molecular biologist; and general preparations, procedures, and considerations for use of the book. The following chapters then discuss cloning vectors and bacterial cells; preparation of DNA from eukaryotic cells; probing nucleic acids; plasmid DNA preparation; DNA restriction fragment preparation; purification of DNA; and preparation and analysis of RNA from eukaryotic cells. Other chapters cover preparation of DNA from bacteriophage clones; cloning DNA from the eukaryotic genome; subcloning into plasmids; M13 cloning and sequencing; further characterization of cloned DNA; transfection of mammalian cells in culture; protein methods; general methods; and specialized methods. This book will be of interest to practitioners in the fields of biology and molecular genetics.
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
- No. of pages: 399
- Language: English
- Edition: 1
- Published: November 15, 2012
- Imprint: Elsevier
- Paperback ISBN: 9780444569462
- eBook ISBN: 9780444601490