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Contents Publisher's Note xxi Chapter 1 Plasmids and Their Usefulness in Molecular Cloning 1.1 1 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation 1. 2 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation 1. 3 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation 1. 4 Preparation of Plasmid DNA by Small-scale Boiling Lysis 1. 5 Preparation of Plasmid DNA by Large-scale Boiling Lysis 1. 6 Preparation of Plasmid DNA: Toothpick Minipreparation 1. 7 Preparation of Plasmid DNA by Lysis with SDS 1. 8 Purification of Plasmid DNA by Precipitation with Polyethylene Glycol 1. 9 Purification of Plasmid DNA by Chromatography 1. 10 Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients 1. 11 Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients 1. 12 Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents 1. 13 Removal of Ethidium Bromide from DNA by Ion-exchange Chromatography 1. 14 Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl 1. 15 Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Chromatography through Sephacryl S-10001. 16 Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride 1. 17 Directional Cloning into Plasmid Vectors 1. 18 Attaching Adaptors to Protruding Termini 1. 19 Blunt-ended Cloning into Plasmid Vectors 1. 20 Dephosphorylation of Plasmid DNA 1. 21 Addition of Synthetic Linkers to Blunt-ended DNA 1. 22 Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose 1. 23 The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation 1. 24 The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra-competent" Cells 1. 25 Preparation and Transformation of Competent E. coli using Calcium Chloride 1. 26 Transformation of E. coli by Electroporation 1. 27 Screening Bacterial Colonies Using X-gal and IPTG: --Complementation 1. 28 Screening Bacterial Colonies by Hybridization: Small Numbers 1. 29 Screening Bacterial Colonies by Hybridization: Intermediate Numbers 1. 30 Screening Bacterial Colonies by Hybridization: Large Numbers 1. 31 Lysing Colonies and Binding of DNA to Filters 1. 32 Hybridization of Bacterial DNA on Filters 1. Chapter 2 Bacteriophage - and Its Vectors 2.1 1 Plating Bacteriophage - 2. 2 Picking Bacteriophage - Plaques 2. 3 Preparing Stocks of Bacteriophage - by Plate Lysis and Elution 2. 4 Preparing Stocks of Bacteriophage - by Small-scale Liquid Culture 2. 5 Large-scale Growth of Bacteriophage -: Infection at Low Multiplicity 2. 6 Precipitation of Bacteriophage - Particles from Large-scale Lysates 2. 7 Assaying the DNA Content of Bacteriophage - Stocks and Lysates by Gel Electrophoresis 2. 8 Purification of Bacteriophage - Particles by Isopycnic Centrifugation through CsCl Gradients 2. 9 Purification of Bacteriophage - Particles by Centrifugation through a Glycerol Step Gradient 2. 10 Purification of Bacteriophage - Particles by Pelleting/Centrifugation 2. 11 Extraction of Bacteriophage - DNA from Large-scale Cultures Using Proteinase K and SDS 2. 12 Extraction of Bacteriophage - DNA from Large-scale Cultures Using Formamide 2. 13 Preparation of Bacteriophage - DNA Cleaved with a Single Restriction Enzyme for Use as a Cloning Vector 2. 14 Preparation of Bacteriophage - DNA Cleaved with Two Restriction Enzymes for Use as a Cloning Vector 2. 15 Alkaline Phosphatase Treatment of Bacteriophage - Vector DNA 2. 16 Purification of Bacteriophage - Arms: Centrifugation through Sucrose Density Gradients 2. 17 Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions 2. 18 Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Preparative Reactions 2. 19 Ligation of Bacteriophage - Arms to Fragments of Foreign Genomic DNA 2. 20 Amplification of Genomic Libraries 2. 21 Transfer of Bacteriophage DNA from Plaques to Filters 2. 22 Hybridization of Bacteriophage DNA on Filters 2. 23 Rapid Analysis of Bacteriophage - Isolates: Purification of - DNA from Plate Lysates 2 24 Rapid Analysis of Bacteriophage - Isolates: Purification of - DNA from Liquid Cultures 2. Chapter 3 Working with Bacteriophage M13 Vectors 3.1 1 Plating Bacteriophage M13 3. 2 Growing Bacteriophage M13 in Liquid Culture 3. 3 Preparation of Double-stranded (Replicative Form) Bacteriophage M13 DNA 3. 4 Preparation of Single-stranded Bacteriophage M13 DNA 3. 5 Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA 3. 6 Cloning into Bacteriophage M13 Vectors 3. 7 Analysis of Recombinant Bacteriophage M13 Clones 3. 8 Producing Single-stranded DNA with Phagemid Vectors 3. Chapter 4 Working with High-capacity Vectors 4.1 1 Construction of Genomic DNA Libraries in Cosmid Vectors 4. 2 Screening an Unamplified Cosmid Library by Hybridization: Plating the Library onto Filters 4. 3 Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture 4. 4 Amplification and Storage of a Cosmid Library: Amplification on Filters 4. 5 Working with Bacteriophage P1 and Its Cloning Systems 4. 6 Transferring P1 Clones between E. coli Hosts 4. 7 Working with Bacterial Artificial Chromosomes 4. 8 Isolation of BAC DNA from Small-scale Cultures 4. 9 Isolation of BAC DNA from Large-scale Cultures 4. 10 Working with Yeast Artificial Chromosomes 4. 11 Growth of S. cerevisiae and Preparation of DNA 4. 12 Small-scale Preparations of Yeast DNA 4. 13 Analyzing Yeast Colonies by PCR 4. 14 Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors: Vectorette Polymerase Chain Reactions 4. Chapter 5 Gel Electrophoresis of DNA and Pulsed-field Agarose 5.1 Gel Electrophoresis 1 Agarose Gel Electrophoresis 5. 2 Detection of DNA in Agarose Gels 5. 3 Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes 5. 4 Recovery of DNA from Agarose and Polyacrylamide Gels: Electroelution into Dialysis Bags 5. 5 Purification of DNA Recovered from Agarose and Polyacrylamide Gels by Anion-exchange Chromatography 5. 6 Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction 5. 7 Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase 5. 8 Alkaline Agarose Gel Electrophoresis 5. 9 Neutral Polyacrylamide Gel Electrophoresis 5. 10 Detection of DNA in Polyacrylamide Gels by Staining 5. 11 Detection of DNA in Polyacrylamide Gels by Autoradiography 5. 12 Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method 5. 13 Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells and Tissues 5. 14 Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast 5. 15 Restriction Endonuclease Digestion of DNA in Agarose Plugs 5. 16 Markers for Pulsed-field Gel Electrophoresis 5. 17 Pulsed-field Gel Electrophoresis via Transverse Alternating Field Electrophoresis Gels 5. 18 Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels 5. 19 Direct Retrieval of DNA Fragments from Pulsed-field Gels 5. 20 Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration 5. Chapter 6 Preparation and Analysis of Eukaryotic Genomic DNA 6.1 1 Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol 6. 2 Isolation of High-molecular-weight DNA from Mammalian Cells Using Formamide 6. 3 Isolation of DNA from Mammalian Cells by Spooling 6. 4 Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates 6. 5 Preparation of Genomic DNA from Mouse Tails and Other Small Samples 6. 6 Rapid Isolation of Mammalian DNA 6. 7 Rapid Isolation of Yeast DNA 6. 8 Southern Blotting: Capillary Transfer of DNA to Membranes 6. 9 Southern Blotting: Simultaneous Transfer of DNA from a Single Agarose Gel to Two Membranes 6. 10 Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes 6. Chapter 7 Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells 7.1 1 Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction 7. 2 A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissues 7. 3 Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography 7. 4 Selection of Poly(A)+ RNA by Batch Chromatography 7. 5 Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels 7. 6 Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde 7. 7 Transfer and Fixation of Denatured RNA to Membranes 7. 8 Northern Hybridization 7. 9 Dot and Slot Hybridization of Purified RNA 7. 10 Mapping RNA with Nuclease S1 7. 11 Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes 7.3 12 Analysis of RNA by Primer Extension 7. Chapter 8 In Vitro Amplification of DNA by the Polymerase Chain Reaction 8.1 1 The Basic Polymerase Chain Reaction 8. 2 Purification of PCR Products in Preparation for Cloning 8. 3 Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration 8. 4 Blunt-end Cloning of PCR Products 8. 5 Cloning PCR Products into T Vectors 8. 6 Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA 8. 7 Genetic Engineering with PCR 8. 8 Amplification of cDNA Generated by Reverse Transcription of mRNA (RT-PCR) 8. 9 Rapid Amplification of 5« cDNA Ends (5«-RACE) 8. 10 Rapid Amplification of 3« cDNA Ends (3«-RACE) 8. 11 Mixed Oligonucleotide-primed Amplification of cDNA (MOPAC) 8. 12 Rapid Characterization of DNAs Cloned in Prokaryotic Vectors 8. 13 Long PCR 8. 14 Inverse PCR 8. 15 Quantitative PCR 8. 16 Differential Display-PCR (DD-PCR) 8. Chapter 9 Preparation of Radiolabeled DNA and RNA Probes 9.1 1 Random Priming: Radiolabeling of Purified DNA Fragments by Extension of Random Oligonucleotides 9. 2 Random Priming: Radiolabeling of DNA by Extension of Random Oligonucleotides in the Presence of Melted Agarose9. 3 Radiolabeling of DNA Probes by the Polymerase Chain Reaction 9. 4 Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates 9. 5 Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates 9. 6 Synthesis of Single-stranded RNA Probes by In Vitro Transcription 9. 7 Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers 9. 8 Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer 9. 9 Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension 9. 10 Labeling 3« Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I 9. 11 Labeling 3« Termini of Double-stranded DNA with Bacteriophage T4 DNA Polymerase 9. 12 End Labeling Protruding 3« Termini of Double-stranded DNA with [--32P]Cordycepin 5« Triphosphate or [--32P]dideoxy ATP 9. 13 Dephosphorylation of DNA Fragments with Alkaline Phosphatase 9. 14 Phosphorylation of DNA Molecules with Protruding 5«-Hydroxyl Termini 9. 15 Phosphorylation of DNA Molecules with Dephosphorylated Blunt Ends or Recessed 5« Termini 9. 16 Phosphorylation of DNA Molecules with Protruding 5« Termini by the Exchange Reaction 9. Chapter 10 Working with Synthetic Oligonucleotide Probes 10.1 1 Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis 10. 2 Phosphorylating the 5« Termini of Oligonucleotides 10. 3 Purification of Radiolabeled Oligonucleotides by Precipitation with Ethanol 10. 4 Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide 10. 5 Purification of Radiolabeled Oligonucleotides by Size-exclusion Chromatography 10. 6 Purification of Radiolabeled Oligonucleotides by Chromatography on a Sep-Pak C18 Column 10. 7 Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I 10. 8 Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts 10. 9 Empirical Measurement of Melting Temperature 10. Chapter 11 Preparation of cDNA Libraries and Gene Identification 11.1 1 Construction of cDNA Libraries 11. Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase 11. Stage 2: Second-strand Synthesis 11. Stage 3: Methylation of cDNA 11. Stage 4: Attachment of Linkers or Adaptors 11. Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose CL-4B 11. Stage 6: Ligation of cDNA to Bacteriophage - Arms 11. 2 Construction and Screening of Eukaryotic Expression Libraries 11. Stage 1: Construction of cDNA Libraries in Eukaryotic Expression Vectors 11. Stage 2: Screening cDNA Libraries Constructed in Eukaryotic Expression Vectors 11. 3 Exon Trapping and Amplification 11. Stage 1: Construction of the Library 11. Stage 2: Electroporation of the Library into COS-7 Cells 11. Stage 3: Harvesting the mRNA 11. Stage 4: Reverse Transcriptase-PCR 11. Stage 5: Analysis of Clones 11. 4 Direct Selection of cDNAs with Large Genomic DNA Clones 11. Chapter 12 DNA Sequencing 12.1 1 Generation of a Library of Randomly Overlapping DNA Inserts 12. 2 Preparing Denatured Double-stranded DNA Templates for Sequencing by Dideoxy-mediated Chain Termination 12. 3 Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase)12. 4 Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I and Single-stranded DNA Templates 12. 5 Dideoxy-mediated Sequencing of DNA Using Taq DNA Polymerase 12. 6 Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers 12. 7 Chemical Sequencing 12. 8 Preparation of Denaturing Polyacrylamide Gels 12. 9 Preparation of Denaturing Polyacrylamide Gels Containing Formamide 12. 10 Preparation of Electrolyte Gradient Gels 12. 11 Loading and Running DNA-sequencing Gels 12. 12 Autoradiography and Reading of Sequencing Gels 12. Chapter 13 Mutagenesis 13.1 1 Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA 13. 2 Oligonucleotide-directed Mutagenesis of Single-stranded DNA 13. 3 In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI 13. 4 Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site USE (Mutagenesis)13. 5 Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method 13. 6 Site-specific Mutagenesis by Overlap Extension 13. 7 Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligonucleotides 13. 8 Detection of Mutations by Single-strand Conformational Polymorphism and Heteroduplex Analysis 13. 9 Generation of Sets of Nested Deletion Mutants with Exonuclease III 13. 10 Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease 13. Chapter 14 Screening Expression Libraries 14.1 1 Screening Expression Libraries Constructed in Bacteriophage - Vectors 14. 2 Screening Expression Libraries Constructed in Plasmid Vectors 14. 3 Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening 14. 4 Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate14. 5 Removal of Cross-reactive Antibodies from Antiserum: Affinity Chromatography 14. 6 Identifying DNA-binding Proteins in Bacteriophage - Expression Libraries 14. 7 Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage -: Lysogens: Lysis of Bacterial Colonies 14. 8 Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage -: Lytic Infections on Agar Plates 14. 9 Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage -: Lytic Infections in Liquid Medium 14. Chapter 15 Expression of Cloned Genes in Escherichia coli 15.1 1 Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters 15. 2 Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter 15. 3 Expression of Cloned Genes in E. coli Using the Bacteriophage - pL Promoter 15. 4 Expression of Secreted Foreign Proteins Using the Alkaline Phosphatase Promoter (phoA) and Signal Sequence 15. 5 Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose15. 6 Purification of Maltose-binding Fusion Proteins by Affinity Chromatography on Amylose Resin 15. 7 Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography 15. 8 Purification of Expressed Proteins from Inclusion Bodies 15. Chapter 16 Introducing Cloned Genes into Cultured Mammalian Cells 16.1 1 DNA Transfection Mediated by Lipofection 16. 2 Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs 16. 3 Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA 16. 4 Transfection Mediated by DEAE-Dextran: High-efficiency Method 16. 5 DNA Transfection by Electroporation 16. 6 DNA Transfection by Biolistics 16. 7 DNA Transfection Using Polybrene 16. Chapter 17 Analysis of Gene Expression in Cultured Mammalian Cells 17.1 1 Mapping Protein-binding Sites on DNA by DNase I Footprinting 17. 2 Gel Retardation Assays for DNA-binding Proteins 17. 3 Mapping DNase-I-hypersensitive Sites 17. 4 Transcriptional Run-on Assays 17. 5 Measurement of Chloramphenicol Acetyltransferase in Extracts of Mammalian Cells Using Thin-layer Chromatography 17. 6 Assay for Luciferase in Extracts of Mammalian Cells 17. 7 Assay for --galactosidase in Extracts of Mammalian Cells 17. 8 Tetracycline as Regulator of Inducible Gene Expression in Mammalian Cells 17. Stage 1: Stable Transfection of Fibroblasts with pTet-tTAk 17. Stage 2: Stable Transfection of Inducible tTA-expressing NIH-3T3 Cells with Tetracycline-regulated Target Genes 17. Stage 3: Analysis of Protein Expression in Transfected Cells 17. 9 Ecdysone as Regulator of Inducible Gene Expression in Mammalian Cells 17. Chapter 18 Protein Interaction Technologies 18.1 1 Two-hybrid and Other Two-component Systems 18. Stage 1: Characterization of a Bait-LexA Fusion Protein 18. Stage 2: Selecting an Interactor 18. Stage 3: Second Confirmation of Positive Interactions 18. 2 Detection of Protein-Protein Interactions Using Far Western with GST Fusion Proteins 18. 3 Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique 18. 4 Identification of Associated Proteins by Coimmunoprecipitation 18. 5 Probing Protein Interactions Using GFP and Fluorescence Resonance Energy Transfer 18. Stage 1: Labeling Proteins with Fluorescent Dyes 18. Stage 2: Cell Preparation for FLIM-FRET Analysis 18. Stage 3: FLIM-FRET Measurements 18. 6 Analysis of Interacting Proteins with Surface Plasmon Resonance Spectroscopy Using BIAcore 18. Stage 1: Preparation of the Capture Surface and Test Binding 18. Stage 2: Kinetic Analysis of the Antibody-Antigen Interaction 18. Appendices 1 Preparation of Reagents and Buffers Used in Molecular Cloning, A1.1 2 Media, A2.1 3 Commonly Used Techniques in Molecular Cloning, A3.1 4 Cautions, A4.1 INDEX, I.1
Library of Congress Subject Headings for this publication:
Molecular cloning -- Laboratory manuals.
Cloning, Molecular -- Laboratory Manuals.