Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

Online ISBN: 9780471142720

DOI: 10.1002/0471142727

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  1. Foreword
  2. Preface
  3. Chapter 1 Escherichia coli, Plasmids, and Bacteriophages
    1. Introduction
    2. Section I Escherichia coli
      1. Unit 1.1 Media Preparation and Bacteriological Tools
      2. Unit 1.2 Growth in Liquid Media
      3. Unit 1.3 Growth on Solid Media
      4. Unit 1.4 Selected Topics from Classical Bacterial Genetics
    3. Section II Vectors Derived from Plasmids
      1. Unit 1.5 Introduction to Plasmid Biology
      2. Unit 1.6 Minipreps of Plasmid DNA
      3. Unit 1.7 Large-Scale Preparation of Plasmid DNA
      4. Unit 1.8 Introduction of Plasmid DNA into Cells
    4. Section III Vectors Derived from Lambda and Related Bacteriophages
      1. Unit 1.9 Introduction to Lambda Phages
      2. Unit 1.10 Lambda as a Cloning Vector
      3. Unit 1.11 Plating Lambda Phage to Generate Plaques
      4. Unit 1.12 Growing Lambda-Derived Vectors
      5. Unit 1.13 Preparing Lambda DNA from Phage Lysates
    5. Section IV Vectors Derived from Filamentous Phages
      1. Unit 1.14 Introduction to Vectors Derived from Filamentous Phages
      2. Unit 1.15 Preparing and Using M13-Derived Vectors
    6. Section V Specialized Techniques
      1. Unit 1.16 Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination
      2. Unit 1.17 E. coli Genome Manipulation by P1 Transduction
  4. Chapter 2 Preparation and Analysis of DNA
    1. Introduction
    2. Section I Manipulation of DNA
      1. Unit 2.1A Purification and Concentration of DNA from Aqueous Solutions
      2. Unit 2.1B Purification of DNA by Anion-Exchange Chromatography
      3. Unit 2.2 Preparation of Genomic DNA from Mammalian Tissue
      4. Unit 2.3 Preparation of Genomic DNA from Plant Tissue
      5. Unit 2.4 Preparation of Genomic DNA from Bacteria
    3. Section II Resolution and Recovery of Large DNA Fragments
      1. Unit 2.5A Agarose Gel Electrophoresis
      2. Unit 2.5B Pulsed-Field Gel Electrophoresis
      3. Unit 2.6 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
    4. Section III Resolution and Recovery of Small DNA Fragments
      1. Unit 2.7 Separation of Small DNA Fragments by Conventional Gel Electrophoresis
      2. Unit 2.8 Capillary Electrophoresis of DNA
    5. Section IV Analysis of DNA Sequences by Blotting and Hybridization
      1. Unit 2.9A Southern Blotting
      2. Unit 2.9B Dot and Slot Blotting of DNA
      3. Unit 2.10 Hybridization Analysis of DNA Blots
    6. Section V Synthesis and Purification of Oligonucleotides
      1. Unit 2.11 Synthesis and Purification of Oligonucleotides
      2. Unit 2.12 Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis
    7. VI Special Protocols for DNA Isolation
      1. Special Protocols for DNA Isolation
      2. Unit 2.13 Preparation of Genomic DNA from Mammalian Sperm
      3. Unit 2.14 DNA Isolation from Mammalian Samples
  5. Chapter 3 Enzymatic Manipulation of DNA and RNA
    1. Introduction
    2. Section I Restriction Endonucleases
      1. Unit 3.1 Digestion of DNA with Restriction Endonucleases
    3. Section II Restriction Mapping
      1. Unit 3.2 Mapping by Multiple Endonuclease Digestions
      2. Unit 3.3 Mapping by Partial Endonuclease Digestions
    4. Section III Enzymes for Modifying and Radioactively Labeling Nucleic Acids
      1. Unit 3.4 Reagents and Radioisotopes Used to Manipulate Nucleic Acids
      2. Unit 3.5 DNA-Dependent DNA Polymerases
      3. Unit 3.6 Template-Independent DNA Polymerases
      4. Unit 3.7 RNA-Dependent DNA Polymerases
      5. Unit 3.8 RNA Polymerases
      6. Unit 3.9 DNA Repair Enzymes
      7. Unit 3.10 Phosphatases and Kinases
      8. Unit 3.11 Exonucleases
      9. Unit 3.12 Endonucleases
      10. Unit 3.13 Ribonucleases
      11. Unit 3.14 DNA Ligases
      12. Unit 3.15 RNA Ligases
    5. Section IV Construction of Hybrid DNA Molecules
      1. Unit 3.16 Subcloning of DNA Fragments
      2. Unit 3.17 Constructing Recombinant DNA Molecules by PCR
    6. Section V Specialized Applications
      1. Unit 3.18 Labeling and Colorimetric Detection of Nonisotopic Probes
      2. Unit 3.19 Chemiluminescent Detection of Nonisotopic Probes
      3. Unit 3.20 Recombinational Cloning
      4. Unit 3.21 DNA Cloning and Engineering by Uracil Excision
      5. Unit 3.22 Gene and Genome Construction in Yeast
      6. Unit 3.23 Design and Assembly of Large Synthetic DNA Constructs
      7. Unit 3.24 Enrichment of Error-Free Synthetic DNA Sequences by CEL I Nuclease
      8. Unit 3.25 Database of DNA Polymerases
  6. Chapter 4 Preparation and Analysis of RNA
    1. Introduction
    2. Section I Preparation of RNA from Eukaryotic and Prokaryotic Cells
      1. Unit 4.1 Preparation of Cytoplasmic RNA from Tissue Culture Cells
      2. Unit 4.2 Guanidine Methods for Total RNA Preparation
      3. Unit 4.3 Phenol/SDS Method for Plant RNA Preparation
      4. Unit 4.4 Preparation of Bacterial RNA
      5. Unit 4.5 Preparation of Poly(A)+ RNA
    3. Section II Analysis of RNA Structure and Synthesis
      1. Unit 4.6 S1 Analysis of Messenger RNA Using Single-Stranded DNA Probes
      2. Unit 4.7 Ribonuclease Protection Assay
      3. Unit 4.8 Primer Extension
      4. Unit 4.9 Analysis of RNA by Northern and Slot Blot Hybridization
      5. Unit 4.10 Identification of Newly Transcribed RNA
      6. Unit 4.11 RNA-Seq: A Method for Comprehensive Transcriptome Analysis
      7. Unit 4.12 Construction of Normalized RNA-seq Libraries for Next-Generation Sequencing Using the Crab Duplex-Specific Nuclease
      8. Unit 4.13 RASL-seq for Massively Parallel and Quantitative Analysis of Gene Expression
      9. Unit 4.14 Native Elongating Transcript Sequencing (NET-seq)
      10. Unit 4.15 Synthesis and Labeling of RNA In Vitro
  7. Chapter 5 Construction of Recombinant DNA Libraries
    1. Introduction
    2. Section I Overview of Recombinant DNA Libraries
      1. Unit 5.1 Genomic DNA Libraries
      2. Unit 5.2 cDNA Libraries
    3. Section II Preparation of Insert DNA from Genomic DNA
      1. Unit 5.3 Size Fractionation Using Sucrose Gradients
      2. Unit 5.4 Size Fractionation Using Agarose Gels
    4. Section III Preparation of Insert DNA from Messenger RNA
      1. Unit 5.5 Conversion of mRNA into Double-Stranded cDNA
      2. Unit 5.6 Ligation of Linkers or Adapters to Double-Stranded cDNA
    5. Section IV Production of Genomic DNA and cDNA Libraries
      1. Unit 5.7 Production of a Genomic DNA Library
      2. Unit 5.8A Production of a Complete cDNA Library
      3. Unit 5.9 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
    6. Section V Amplification of Transformed or Packaged Libraries
      1. Unit 5.10 Amplification of a Bacteriophage Library
      2. Unit 5.11 Amplification of Cosmid and Plasmid Libraries
      3. Unit 5.12 Normalizing cDNA Libraries
  8. Chapter 6 Screening of Recombinant DNA Libraries
    1. Introduction
    2. Section I Plating Libraries and Transfer to Filter Membranes
      1. Unit 6.1 Plating and Transferring Bacteriophage Libraries
      2. Unit 6.2 Plating and Transferring Cosmid and Plasmid Libraries
    3. Section II Hybridization with Radioactive Probes
      1. Unit 6.3 Using DNA Fragments as Probes
      2. Unit 6.4 Using Synthetic Oligonucleotides as Probes
    4. Section III Purification of Bacteriophage, Cosmid, and Plasmid Clones
      1. Unit 6.5 Purification of Bacteriophage Clones
      2. Unit 6.6 Purification of Cosmid and Plasmid Clones
    5. Section IV Screening with Antibodies
      1. Unit 6.7 Immunoscreening of Fusion Proteins Produced in Lambda Plaques
      2. Unit 6.8 Immunoscreening after Hybrid Selection and Translation
    6. Section V Yeast Artificial Chromosome Libraries
      1. Unit 6.9 Overview of Strategies for Screening YAC Libraries and Analyzing YAC Clones
      2. Unit 6.10 Analysis of Isolated YAC Clones
    7. Section VI Specialized Strategies for Screening Libraries
      1. Unit 6.11 Use of Monoclonal Antibodies for Expression Cloning
      2. Unit 6.12 Recombination-Based Assay (RBA) for Screening Bacteriophage Lambda Libraries
  9. Chapter 7 DNA Sequencing
    1. Introduction
    2. Unit 7.1 Overview of DNA Sequencing Strategies
    3. Unit 7.2 “First Generation” Automated DNA Sequencing Technology
    4. Unit 7.3 Preparation of Templates for DNA Sequencing
    5. Unit 7.4A DNA Sequencing by the Dideoxy Method
    6. Unit 7.4B Dideoxy DNA Sequencing with Chemiluminescent Detection
    7. Unit 7.5 High-Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems
    8. Unit 7.6 Denaturing Gel Electrophoresis for Sequencing
    9. Unit 7.7 Computer Manipulation of DNA and Protein Sequences
    10. Unit 7.8 Polony DNA Sequencing
    11. Unit 7.9 Bisulfite Sequencing of DNA
    12. Unit 7.10 Single Molecule Sequencing with a HeliScope Genetic Analysis System
    13. Unit 7.11 Multiplex Illumina Sequencing Using DNA Barcoding
    14. Unit 7.12 Exome Sequencing by Targeted Enrichment
  10. Chapter 8 Mutagenesis of Cloned DNA
    1. Introduction
    2. Unit 8.1 Oligonucleotide-Directed Mutagenesis without Phenotypic Selection
    3. Unit 8.2A Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence
    4. Unit 8.2B Gene Synthesis: Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides
    5. Unit 8.3 Random Mutagenesis by PCR
    6. Unit 8.4 Linker-Scanning Mutagenesis of DNA
    7. Unit 8.5 Directed Mutagenesis Using the Polymerase Chain Reaction
  11. Chapter 9 Introduction of DNA into Mammalian Cells
    1. Introduction
    2. Section I Transfection of DNA into Eukaryotic Cells
      1. Unit 9.1 Calcium Phosphate Transfection
      2. Unit 9.2 Transfection Using DEAE-Dextran
      3. Unit 9.3 Transfection by Electroporation
      4. Unit 9.4 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
      5. Unit 9.5 Selection of Transfected Mammalian Cells
    3. Section II Uses of Fusion Genes in Mammalian Transfection
      1. Unit 9.6 Overview of Genetic Reporter Systems
      2. Unit 9.7A Isotopic Assays for Reporter Gene Activity
      3. Unit 9.7B Nonisotopic Assays for Reporter Gene Activity
      4. Unit 9.7C Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics in Vivo
      5. Unit 9.8 Direct Analysis of RNA after Transfection
    4. Section III Transduction of Genes Using Retrovirus Vectors
      1. Unit 9.9 Overview of the Retrovirus Transduction System
      2. Unit 9.10 Preparation of a Specific Retrovirus Producer Cell Line
      3. Unit 9.11 Transient Transfection Methods for Preparation of High-Titer Retroviral Supernatants
      4. Unit 9.12 Large-Scale Preparation and Concentration of Retrovirus Stocks
      5. Unit 9.13 Detection of Helper Virus in Retrovirus Stocks
      6. Unit 9.14 Retrovirus Infection of Cells In Vitro and In Vivo
    5. Section IV Inactivation of Genes in Mammalian Cells
      1. Unit 9.15 Human Somatic Cell Gene Targeting
  12. Chapter 10 Analysis of Proteins
    1. Introduction
    2. Section I Quantitation of Proteins
      1. Unit 10.1A Spectrophotometric and Colorimetric Determination of Protein Concentration
      2. Unit 10.1B Quantitative Amino Acid Analysis
    3. Section II Electrophoretic Separation of Proteins
      1. Unit 10.2A One-Dimensional SDS Gel Electrophoresis of Proteins
      2. Unit 10.2B One-Dimensional Electrophoresis Using Nondenaturing Conditions
      3. Unit 10.3 Two-Dimensional Gel Electrophoresis Using the ISO-DALT System
      4. Unit 10.4 Two-Dimensional Gel Electrophoresis
      5. Unit 10.5 Overview of Digital Electrophoresis Analysis
    4. III Detection of Proteins
      1. III Detection of Proteins
      2. Unit 10.6 Staining Proteins in Gels
      3. Unit 10.7 Detection of Proteins on Blot Transfer Membranes
      4. Unit 10.8 Immunoblotting and Immunodetection
    5. Section IV Purification of Proteins by Conventional Chromatography
      1. Unit 10.9 Gel-Filtration Chromatography
      2. Unit 10.10 Ion-Exchange Chromatography
      3. Unit 10.11A Immunoaffinity Chromatography
      4. Unit 10.11B Metal-Chelate Affinity Chromatography
    6. Section V Purification of Proteins by High-Performance Liquid Chromatography
      1. Unit 10.12 HPLC of Peptides and Proteins: Preparation and System Set-Up
      2. Unit 10.13 HPLC of Peptides and Proteins: Standard Operating Conditions
      3. Unit 10.14 Reversed-Phase Isolation of Peptides
    7. Section VI Specialized Applications
      1. Unit 10.15 Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging
      2. Unit 10.16 Immunoprecipitation
      3. Unit 10.18 Metabolic Labeling with Amino Acids
      4. Unit 10.19 Isolation of Proteins for Microsequence Analysis
      5. Unit 10.20 Capillary Electrophoresis of Proteins and Peptides
      6. Unit 10.21 Overview of Peptide and Protein Analysis by Mass Spectrometry
      7. Unit 10.22 Protein Identification and Characterization by Mass Spectrometry
      8. Unit 10.23 Difference Gel Electrophoresis (DIGE) Using CyDye DIGE Fluor Minimal Dyes
      9. Unit 10.24 Solution Radioimmunoassay of Proteins and Peptides
      10. Unit 10.25 Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow
      11. Unit 10.26 Isolation of Proteins by Heat-Induced Extraction from Formalin-Fixed, Paraffin-Embedded Tissue and Preparation of Tryptic Peptides for Mass Spectrometric Analysis
      12. Unit 10.27 Extraction of Proteins from Formalin-Fixed, Paraffin-Embedded Tissue Using the Qproteome Extraction Technique and Preparation of Tryptic Peptides for Liquid Chromatography/Mass Spectrometry Analysis
      13. Unit 10.28 The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer Optimization as an Aid in Protein Crystallization
      14. Unit 10.29 Biochemical Analysis of Protein SUMOylation
      15. Unit 10.30 Assays for Investigating deSUMOylation Enzymes
  13. Chapter 11 Immunology
    1. Introduction
    2. Section I Immunoassays
      1. Unit 11.1 Conjugation of Enzymes to Antibodies
      2. Unit 11.2 Enzyme-Linked Immunosorbent Assays (ELISA)
      3. Unit 11.3 Isotype Determination of Antibodies
    3. Section II Preparation of Monoclonal Antibodies
      1. Unit 11.4 Immunization of Mice
      2. Unit 11.5 Preparation of Myeloma Cells
      3. Unit 11.6 Preparation of Mouse Feeder Cells for Fusion and Cloning
      4. Unit 11.7 Fusion of Myeloma Cells with Immune Spleen Cells
      5. Unit 11.8 Cloning of Hybridoma Cell Lines by Limiting Dilution
      6. Unit 11.9 Freezing and Recovery of Hybridoma Cell Lines
      7. Unit 11.10 Production of Monoclonal Antibody Supernatant and Ascites Fluid
      8. Unit 11.11 Purification of Monoclonal Antibodies
    4. Section III Preparation of Polyclonal Antisera
      1. Unit 11.12 Production of Polyclonal Antisera
      2. Unit 11.13 In Vitro Antibody Production
      3. Unit 11.14 Purification of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or Hybridoma Supernatant
    5. Section IV Preparation of Antipeptide Antibodies
      1. Unit 11.15 Introduction to Peptide Synthesis
      2. Unit 11.16 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
    6. Section V Determination of Specific Antibody Titer and Isotype
      1. Unit 11.17 Determination of the Specific Antibody Titer
    7. Section VI Preparation and Use of Specialized Antibodies
      1. Unit 11.18 Identification of Polyol-Responsive Monoclonal Antibodies for Use in Immunoaffinity Chromatography
  14. Chapter 12 DNA-Protein Interactions
    1. Introduction
    2. Unit 12.1 Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
    3. Unit 12.2 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
    4. Unit 12.3 Methylation and Uracil Interference Assays for Analysis of Protein-DNA Interactions
    5. Unit 12.4 DNase I Footprint Analysis of Protein-DNA Binding
    6. Unit 12.5 UV Crosslinking of Proteins to Nucleic Acids
    7. Unit 12.6 Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems
    8. Unit 12.7 Detection, Purification, and Characterization of cDNA Clones Encoding DNA-Binding Proteins
    9. Unit 12.8 Rapid Separation of Protein-Bound DNA from Free DNA Using Nitrocellulose Filters
    10. Unit 12.9 Analysis of DNA-Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes
    11. Unit 12.10 Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography
    12. Unit 12.11 Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection
    13. Unit 12.12 Yeast One-Hybrid Screening for DNA-Protein Interactions
    14. Unit 12.13 Engineering Designer Nucleases with Customized Cleavage Specificities
    15. Unit 12.14 In Vitro Transcription and Immobilized Template Analysis of Preinitiation Complexes
    16. Unit 12.15 Engineering Designer Transcription Activator--Like Effector Nucleases (TALENs) by REAL or REAL-Fast Assembly
  15. Chapter 13 Yeast
    1. Introduction
    2. Section I Basic Techniques of Yeast Genetics
      1. Unit 13.1 Preparation of Yeast Media
      2. Unit 13.2 Growth and Manipulation of Yeast
      3. Unit 13.2B Drug-Induced Permeabilization of S. cerevisiae
      4. Unit 13.3 Genome-Wide Transposon Mutagenesis in Yeast
      5. Unit 13.3B EMS and UV Mutagenesis in Yeast
    3. Section II Yeast Vectors
      1. Unit 13.4 Yeast Cloning Vectors and Genes
      2. Unit 13.6 Yeast Vectors and Assays for Expression of Cloned Genes
    4. Section III Manipulation of Yeast Genes
      1. Unit 13.7 Introduction of DNA into Yeast Cells
      2. Unit 13.8 Cloning Yeast Genes by Complementation
      3. Unit 13.9 Manipulation of Plasmids from Yeast Cells
      4. Unit 13.10 Manipulation of Cloned Yeast DNA
      5. Unit 13.10B Conditional Depletion of Nuclear Proteins by the Anchor Away System
    5. Section IV Preparation of Yeast DNA, RNA, and Proteins
      1. Unit 13.11 Preparation of Yeast DNA
      2. Unit 13.12 Preparation of Yeast RNA
      3. Unit 13.13 Preparation of Protein Extracts from Yeast
    6. Section V Schizosaccharomyces pombe
      1. Unit 13.14 Overview of Schizosaccharomyces pombe
      2. Unit 13.15 S. pombe Strain Maintenance and Media
      3. Unit 13.16 Growth and Manipulation of S. pombe
      4. Unit 13.17 Introduction of DNA into S. pombe Cells
  16. Chapter 14 In Situ Hybridization and Immunohistochemistry
    1. Introduction
    2. Unit 14.1 Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
    3. Unit 14.2 Cryosectioning
    4. Unit 14.3 In Situ Hybridization to Cellular RNA
    5. Unit 14.4 Detection of Hybridized Probe
    6. Unit 14.5 Counterstaining and Mounting of Autoradiographed In Situ Hybridization Slides
    7. Unit 14.6 Immunohistochemistry
    8. Unit 14.7 In Situ Hybridization and Detection Using Nonisotopic Probes
    9. Unit 14.8 In Situ Polymerase Chain Reaction and Hybridization to Detect Low-Abundance Nucleic Acid Targets
    10. Unit 14.9 Whole-Mount In Situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs
    11. Unit 14.10 Principles and Application of Fluorescence Microscopy
    12. Unit 14.11 Basic Confocal Microscopy
    13. Unit 14.12 Measurement of In Situ Hybridization
    14. Unit 14.13 Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis
    15. Unit 14.14 Whole-Mount Histochemical Detection of beta-Galactosidase Activity
    16. Unit 14.15 Basic Image Analysis and Manipulation in ImageJ
    17. Unit 14.16 Three-Dimensional Reconstruction of Tissues
    18. Unit 14.17 Using CellProfiler for Automatic Identification and Measurement of Biological Objects in Images
    19. Unit 14.18 Using Cell-ID 1.4 with R for Microscope-Based Cytometry
    20. Unit 14.19 Visualization of Microscopy-Based Spectral Imaging Data from Multi-Label Tissue Sections
    21. Unit 14.20 Computer Control of Microscopes Using µManager
    22. Unit 14.21 Use of YouScope to Implement Systematic Microscopy Protocols
    23. Unit 14.22 Using CellX to Quantify Intracellular Events
  17. Chapter 15 The Polymerase Chain Reaction
    1. Introduction
    2. Unit 15.1 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
    3. Unit 15.2 Direct DNA Sequencing of PCR Products
    4. Unit 15.3 Ligation-Mediated PCR for Genomic Sequencing and Footprinting
    5. Unit 15.4 Molecular Cloning of PCR Products
    6. Unit 15.5 Enzymatic Amplification of RNA by PCR (RT-PCR)
    7. Unit 15.6 cDNA Amplification Using One-Sided (Anchored) PCR
    8. Unit 15.7 Quantitation of Rare DNAs by PCR
    9. Unit 15.8 High-Throughput Real-Time Quantitative Reverse Transcription PCR
    10. Unit 15.9 Heat-Activatable Primers for Hot-Start PCR and Hot-Start One-Step RT-PCR: Endpoint and Real-Time Experiments
    11. Unit 15.10 Stem-Loop RT-qPCR for miRNAs
  18. Chapter 16 Protein Expression
    1. Introduction
    2. Section I Expression of Proteins in Escherichia coli
      1. Unit 16.1 Overview of Protein Expression in E. coli
      2. Unit 16.2 Expression Using the T7 RNA Polymerase/Promoter System
      3. Unit 16.3 Expression Using Vectors with Phage lambda Regulatory Sequences
      4. Unit 16.4A Introduction to Expression by Fusion Protein Vectors
      5. Unit 16.4B Enzymatic and Chemical Cleavage of Fusion Proteins
      6. Unit 16.5 Expression and Purification of lacZ and trpE Fusion Proteins
      7. Unit 16.6 Expression and Purification of Maltose-Binding Protein Fusions
      8. Unit 16.7 Expression and Purification of Glutathione-S-Transferase Fusion Proteins
      9. Unit 16.8 Expression and Purification of Thioredoxin Fusion Proteins
    3. Section II Expression of Proteins in Insect Cells Using Baculovirus Vectors
      1. Unit 16.9 Overview of the Baculovirus Expression System
      2. Unit 16.10 Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses
      3. Unit 16.11 Expression and Purification of Recombinant Proteins Using the Baculovirus System
    4. Section III Expression of Proteins in Mammalian Cells
      1. Unit 16.12 Transient Expression of Proteins Using COS Cells
      2. Unit 16.13 Expression and Purification of Epitope-Tagged Multisubunit Protein Complexes from Mammalian Cells
      3. Unit 16.14 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
      4. Unit 16.15 Overview of the Vaccinia Virus Expression System
      5. Unit 16.16 Preparation of Cell Cultures and Vaccinia Virus Stocks
      6. Unit 16.17 Generation of Recombinant Vaccinia Viruses
      7. Unit 16.18 Characterization of Recombinant Vaccinia Viruses and Their Products
      8. Unit 16.19 Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase Hybrid System
      9. Unit 16.20 Expression of Proteins Using Semliki Forest Virus Vectors
      10. Unit 16.21 Overview of the HIV-1 Lentiviral Vector System
      11. Unit 16.22 Generation of HIV-1-Based Lentiviral Vector Particles
      12. Unit 16.23 Amplification Using CHO Cell Expression Vectors
      13. Unit 16.24 Helper-Dependent Adenoviral Vectors
      14. Unit 16.25 Production of Recombinant Adeno-Associated Viral Vectors for In Vitro and In Vivo Use
  19. Chapter 17 Preparation and Analysis of Glycoconjugates
    1. Introduction
    2. Section I Special Considerations for Glycoproteins and Their Purification
      1. Unit 17.1 Special Considerations for Glycoproteins and Their Purification
      2. Unit 17.2 Special Considerations for Proteoglycans and Glycosaminoglycans and Their Purification
      3. Unit 17.3 Special Considerations for Glycolipids and Their Purification
    3. II Detection of Saccharides on Glycoconjugates
      1. II Detection of Saccharides on Glycoconjugates
      2. Unit 17.4 Metabolic Radiolabeling of Animal Cell Glycoconjugates
      3. Unit 17.5 Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction
      4. Unit 17.6 Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine
      5. Unit 17.7 Lectin Analysis of Proteins Blotted onto Filters
      6. Unit 17.8 Detection of Glycophospholipid Anchors on Proteins
      7. Unit 17.9 Direct Chemical Analysis of Glycoconjugates for Carbohydrates
      8. Unit 17.10A Inhibition of N-Linked Glycosylation
      9. Unit 17.10B Inhibition of Glycolipid Biosynthesis
      10. Unit 17.11 Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly
    4. Section III Release of Saccharides from Glycoconjugates
      1. Unit 17.12 Sialidases
      2. Unit 17.13A Endoglycosidase and Glycoamidase Release of N-Linked Glycans
      3. Unit 17.13B Analysis of Glycosaminoglycans with Polysaccharide Lyases
      4. Unit 17.14A Preparation of Glycopeptides
      5. Unit 17.14B Detection of Individual Glycosylation Sites on Glycoproteins
      6. Unit 17.15A beta-Elimination for Release of O-Linked Glycosaminoglycans from Proteoglycans
      7. Unit 17.15B beta-Elimination for Release of O-GalNAc-Linked Oligosaccharides from Glycoproteins and Glycopeptides
      8. Unit 17.16 Acid Hydrolysis for Release of Monosaccharides
      9. Unit 17.17A Enzymatic Release of Oligosaccharides from Glycolipids
      10. Unit 17.17B Endo-beta-Galactosidases and Keratanase
    5. Section IV Analysis of Saccharides Released from Glycoconjugates
      1. Unit 17.18 Analysis of Monosaccharides
      2. Unit 17.19A Total Compositional Analysis by High-Performance Liquid Chromatography or Gas-Liquid Chromatography
      3. Unit 17.19B Composition of Labeled Monosaccharides from Glycosaminoglycans
      4. Unit 17.20 Analysis of Oligosaccharide Negative Charge by Anion-Exchange Chromatography
      5. Unit 17.21A HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides
      6. Unit 17.21B Fractionation and Analysis of Neutral Oligosaccharides by HPLC
      7. Unit 17.22A Nitrous Acid Degradation of Glycosaminoglycans
      8. Unit 17.22B Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans
      9. Unit 17.23 Analysis of Sulfate Esters by Solvolysis or Hydrolysis
  20. Chapter 18 Analysis of Protein Phosphorylation
    1. Introduction
    2. Unit 18.1 Overview of Protein Phosphorylation
    3. Unit 18.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
    4. Unit 18.3 Phosphoamino Acid Analysis
    5. Unit 18.4 Analysis of Phosphorylation of Unlabeled Proteins
    6. Unit 18.5 Detection of Phosphorylation by Enzymatic Techniques
    7. Unit 18.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
    8. Unit 18.7 Assays of Protein Kinases Using Exogenous Substrates
    9. Unit 18.8 Permeabilization Strategies to Study Protein Phosphorylation
    10. Unit 18.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites
    11. Unit 18.10 Use of Protein Phosphatase Inhibitors
    12. Unit 18.11 Design and Use of Analog-Sensitive Protein Kinases
    13. Unit 18.12 The Detection of MAPK Signaling
    14. Unit 18.13 Isolation of Phosphopeptides by Immobilized Metal Ion Affinity Chromatography
    15. Unit 18.14 Analysis of Serine-Threonine Kinase Specificity Using Arrayed Positional Scanning Peptide Libraries
    16. Unit 18.15 Visualization of Kinase Activity with FRET-Based Activity Biosensors
    17. Unit 18.16 Analysis of Protein Tyrosine Phosphatases and Substrates
    18. Unit 18.17 Fluorescent Peptide Assays for Protein Kinases
    19. Unit 18.18 Serine/Threonine Protein Phosphatase Assays
    20. Unit 18.19 Determining In Vivo Phosphorylation Sites Using Mass Spectrometry
    21. Unit 18.20 Using Phospho-Motif Antibodies to Determine Kinase Substrates
  21. Chapter 19 Informatics for Molecular Biologists
    1. Introduction
    2. Unit 19.1 Detecting the Signatures of Adaptive Evolution in Protein-Coding Genes
    3. Unit 19.2 Sequence Databases: Integrated Information Retrieval and Data Submission
    4. Unit 19.3 Sequence Similarity Searching Using the BLAST Family of Programs
    5. Unit 19.4 Protein Databases on the Internet
    6. Unit 19.5 Basic Protein Sequence Analysis
    7. Unit 19.6 Analysis and Management of Microarray Gene Expression Data
    8. Unit 19.7 Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries
    9. Unit 19.8 Resources for Small Regulatory RNAs
    10. Unit 19.9 The UCSC Genome Browser: What Every Molecular Biologist Should Know
    11. Unit 19.10 Galaxy: A Web-Based Genome Analysis Tool for Experimentalists
    12. Unit 19.11 Phylogenetic Analysis of Protein Sequence Data Using the Randomized Axelerated Maximum Likelihood (raxml) Program
  22. Chapter 20 Analysis of Protein Interactions
    1. Introduction
    2. Unit 20.1 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
    3. Unit 20.2 Affinity Purification of Proteins Binding to GST Fusion Proteins
    4. Unit 20.3 Phage-Based Expression Cloning to Identify Interacting Proteins
    5. Unit 20.4 Surface Plasmon Resonance for Measurements of Biological Interest
    6. Unit 20.5 Detection of Protein-Protein Interactions by Coprecipitation
    7. Unit 20.6 Identification of Protein Interactions by Far Western Analysis
    8. Unit 20.7 Two-Hybrid Dual Bait System
    9. Unit 20.8 Interaction Trap/Two-Hybrid System to Identify Loss-of-Interaction Mutant Proteins
  23. Chapter 21 Chromatin Assembly and Analysis
    1. Introduction
    2. Unit 21.1 Micrococcal Nuclease Analysis of Chromatin Structure
    3. Unit 21.2 Separation of Histone Variants and Post-Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis
    4. Unit 21.3 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo
    5. Unit 21.4 DNase I and Hydroxyl Radical Characterization of Chromatin Complexes
    6. Unit 21.5 Isolation of Histones and Nucleosome Cores from Mammalian Cells
    7. Unit 21.6 Assembly of Nucleosomal Templates by Salt Dialysis
    8. Unit 21.7 Chromatin Assembly Using Drosophila Systems
    9. Unit 21.8 Analysis of Protein Co-Occupancy by Quantitative Sequential Chromatin Immunoprecipitation
    10. Unit 21.9 Defining In Vivo Targets of Nuclear Proteins by Chromatin Immunoprecipitation and Microarray Analysis
    11. Unit 21.10 Identifying Chromosomal Targets of DNA-Binding Proteins by Sequence Tag Analysis of Genomic Enrichment (STAGE)
    12. Unit 21.11 Mapping Chromatin Interactions by Chromosome Conformation Capture
    13. Unit 21.12 Paired-End diTagging for Transcriptome and Genome Analysis
    14. Unit 21.13 ChIP-chip for Genome-Wide Analysis of Protein Binding in Mammalian Cells
    15. Unit 21.14 Chromosome Conformation Capture Carbon Copy Technology
    16. Unit 21.15 Chromatin Interaction Analysis Using Paired-End Tag Sequencing
    17. Unit 21.16 Mapping Networks of Protein-Mediated Physical Interactions Between Chromatin Elements
    18. Unit 21.17 Methylation-Sensitive Single-Molecule Analysis of Chromatin Structure
    19. Unit 21.18 Installation of Site-Specific Methylation into Histones Using Methyl Lysine Analogs
    20. Unit 21.19 ChIP-Seq: A Method for Global Identification of Regulatory Elements in the Genome
    21. Unit 21.20 Genome-Wide Location Analysis by Pull Down of In Vivo Biotinylated Transcription Factors
    22. Unit 21.21 DamIP: Using Mutant DNA Adenine Methyltransferase to Study DNA-Protein Interactions In Vivo
    23. Unit 21.22 Simultaneous Single-Molecule Mapping of Protein-DNA Interactions and DNA Methylation by MAPit
    24. Unit 21.23 Single Nucleotide Analysis of Cytosine Methylation by Whole-Genome Shotgun Bisulfite Sequencing
    25. Unit 21.24 ChIP-exo Method for Identifying Genomic Location of DNA-Binding Proteins with Near-Single-Nucleotide Accuracy
    26. Unit 21.25 Capture Hybridization Analysis of RNA Targets (CHART)
    27. Unit 21.26 A Detailed Protocol for Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)
  24. Chapter 22 Nucleic Acid Arrays
    1. Introduction
    2. Unit 22.1 Overview of DNA Microarrays: Types, Applications, and Their Future
    3. Unit 22.2 Preparation of mRNA for Expression Monitoring
    4. Unit 22.3 Profiling Human Gene Expression with cDNA Microarrays
    5. Unit 22.4 Overview of mRNA Expression Profiling Using DNA Microarrays
    6. Unit 22.5 Pattern Discovery in Expression Profiling Data
  25. Chapter 23 Manipulating the Mouse Genome
    1. Introduction
    2. Unit 23.1 Overview of Gene Targeting by Homologous Recombination
    3. Unit 23.2 Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation
    4. Unit 23.3 Mouse Embryonic Stem (ES) Cell Culture
    5. Unit 23.4 Mouse Embryonic Stem (ES) Cell Isolation
    6. Unit 23.5 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
    7. Unit 23.6 Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)
    8. Unit 23.7 Chimeric Mouse Production by Microinjection
    9. Unit 23.8 Mouse Colony Management
    10. Unit 23.9 Transgenic Mouse Production By Zygote Injection
    11. Unit 23.10 Transgenic Mouse Colony Management
    12. Unit 23.11 Modification and Production of BAC Transgenes
    13. Unit 23.12 Regulation of Transgene Expression Using Tetracycline
    14. Unit 23.13 Recombineering-Based Procedure for Creating Cre/loxP Conditional Knockouts in the Mouse
    15. Unit 23.14 Recombineering-Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines
    16. Unit 23.15 Reprogramming of Mouse, Rat, Pig, and Human Fibroblasts into iPS Cells
  26. Chapter 24 Generation and Use of Combinatorial Libraries
    1. Unit 24.1 Overview of Receptors from Combinatorial Nucleic Acid and Protein Libraries
    2. Unit 24.2 Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection
    3. Unit 24.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
    4. Unit 24.4 Peptide Aptamers: Dominant “Genetic” Agents for Forward and Reverse Analysis of Cellular Processes
    5. Unit 24.5 Protein Selection Using mRNA Display
    6. Unit 24.6 Directed Evolution of Proteins In Vitro Using Compartmentalization in Emulsions
  27. Chapter 25 Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell Populations
    1. Introduction
    2. Section A Nucleic Acid Amplification from Individual Cells
      1. Unit 25A.1 Laser Capture Microdissection
      2. Unit 25A.2 Preparation of Single Cells from Solid Tissues for Analysis by PCR
      3. Unit 25A.3 Laser Microdissection-Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA
    3. Section B Molecular Methods for Discovery of Differentially Expressed Genes
      1. Unit 25B.1 Production of a Subtracted cDNA Library
      2. Unit 25B.2 PCR-Based Subtractive cDNA Cloning
      3. Unit 25B.3 Differential Display of mRNA by PCR
      4. Unit 25B.4 Restriction-Mediated Differential Display (RMDD)
      5. Unit 25B.5 AFLP-Based Transcript Profiling
      6. Unit 25B.6 Serial Analysis of Gene Expression (SAGE): Experimental Method and Data Analysis
      7. Unit 25B.7 Representational Difference Analysis
      8. Unit 25B.8 Gene Expression Analysis of a Single or Few Cells
      9. Unit 25B.9 Quantification of Gene Transcripts with Deep Sequencing Analysis of Gene Expression (DSAGE) Using 1 to 2 µg Total RNA
      10. Unit 25B.10 Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System
  28. Chapter 26 Gene Silencing
    1. Introduction
    2. Unit 26.1 Overview of RNA Interference and Related Processes
    3. Unit 26.2 Gene Silencing by RNAi in Mammalian Cells
    4. Unit 26.3 RNA Interference in Caenorhabditis Elegans
    5. Unit 26.4 Cloning of Small RNA Molecules
    6. Unit 26.5 RNA Interference in Cultured Drosophila Cells
    7. Unit 26.6 RNAi in Transgenic Plants
    8. Unit 26.7 Analysis of Small Endogenous RNAs
    9. Unit 26.8 Using Morpholinos to Control Gene Expression
  29. Chapter 27 RNA-Protein Interactions
    1. Introduction
    2. Unit 27.1 Agarose Gel Separation/Isolation of RNA-Protein Complexes
    3. Unit 27.2 Identification of RNA Binding Proteins by UV Cross-Linking
    4. Unit 27.3 Purification of Functional RNA-Protein Complexes using MS2-MBP
    5. Unit 27.4 RNA Immunoprecipitation for Determining RNA-Protein Associations In Vivo
  30. Chapter 28 Mammalian Cell Culture
    1. Introduction
    2. Unit 28.1 Preparation, Culture, and Immortalization of Mouse Embryonic Fibroblasts
    3. Unit 28.2 Isolation and Immortalization of Lymphocytes
    4. Unit 28.3 Establishment and Culture of Human Skin Fibroblasts
  31. Chapter 29 Mouse Phenotyping
    1. Introduction
    2. Section A General Considerations in Mouse Phenotyping
      1. Unit 29A.1 Uses of Forward and Reverse Genetics in Mice to Study Gene Function
      2. Unit 29A.2 Minimizing Variation Due to Genotype and Environment
      3. Unit 29A.3 Collection of Blood and Plasma from the Mouse
      4. Unit 29A.4 Tissue Collection for Systematic Phenotyping in the Mouse
    3. Section B Metabolic Exploration of the Mouse
      1. Unit 29B.1 Evaluation of Energy Homeostasis
      2. Unit 29B.2 Lipid and Bile Acid Analysis
      3. Unit 29B.3 Evaluation of Glucose Homeostasis
      4. Unit 29B.4 Histopathology in Mouse Metabolic Investigations
      5. Unit 29B.5 Dietary Manipulation of Mouse Metabolism
  32. Chapter 30 Metabolomics
    1. Introduction
    2. Unit 30.1 Untargeted Metabolomics
    3. Unit 30.2 Targeted Metabolomics
  33. Appendix 1 Standard Measurements, Data, and Abbreviations
    1. 1A Common Abbreviations
    2. 1B Useful Measurements and Data
    3. 1C Characteristics of Amino Acids
    4. 1D Characteristics of Nucleic Acids
    5. 1E Radioactivity
    6. 1F Safe Use of Radioisotopes
    7. 1G Centrifuges and Rotors
    8. 1H Safe Use of Hazardous Chemicals
    9. 1I Commonly Used Detergents
    10. 1J Common Conversion Factors
    11. 1K Compendium of Drugs Commonly Used in Molecular Biology Research
  34. Appendix 2 Commonly Used Reagents and Equipment
    1. 2 Commonly Used Reagents and Equipment
  35. Appendix 3 Commonly Used Techniques in Biochemistry and Molecular Biology
    1. 3A Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
    2. 3B Silanizing Glassware
    3. 3C Dialysis and Ultrafiltration
    4. 3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
    5. 3E Introduction of Restriction Enzyme Recognition Sequences by Silent Mutation
    6. 3F Techniques for Mammalian Cell Tissue Culture
    7. 3G Importing Biological Materials
    8. 3H Kinetic Assay Methods
    9. 3I Statistics for the Molecular Biologist: Group Comparisons
    10. 3J Microvolume Quantitation of Nucleic Acids
  36. Appendix 4 Suppliers
    1. 4 Selected Suppliers of Reagents and Equipment
  37. Appendix 5 Vectors
    1. 5 Vectors
  38. Appendix 6 Pathway Modulators and Inhibitors
    1. 6 Pathway Modulators and Inhibitors

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