
Basic Methods in Molecular Biology
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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 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.
Table of Contents
Foreword
Acknowledgments
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 Strains
5. 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 Probe
7. 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 Clones
17. 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
Product details
- No. of pages: 399
- Language: English
- Copyright: © Elsevier 1986
- Published: January 1, 1986
- Imprint: Elsevier
- eBook ISBN: 9780444601490
About the Author
Leonard Davis
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