Basic Methods in Molecular Biology

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.

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