Basic Methods in Molecular Biology

Basic Methods in Molecular Biology

1st Edition - January 1, 1986

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  • Author: Leonard Davis
  • eBook ISBN: 9780444601490

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Description

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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