Current Protocols in Molecular Biology

Last Update: June 28, 2010

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Print ISSN: 1934-3639
Online ISSN: 1934-3647
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 Overview
 Table of Contents
 New Protocols
 Sample Unit
 Editors & Contributors
TABLE OF CONTENTS

 Contributors
 Foreword
 Preface
 Chapter 1 Escherichia coli, Plasmids, and Bacteriophages
 Introduction
 Section I Escherichia coli
 UNIT 1.1 Media Preparation and Bacteriological Tools
 UNIT 1.2 Growth in Liquid Media
 UNIT 1.3 Growth on Solid Media
 UNIT 1.4 Selected Topics from Classical Bacterial Genetics
 Section II Vectors Derived from Plasmids
 UNIT 1.5 Introduction to Plasmid Biology
 UNIT 1.6 Minipreps of Plasmid DNA
 UNIT 1.7 Large‐Scale Preparation of Plasmid DNA
 UNIT 1.8 Introduction of Plasmid DNA into Cells
 Section III Vectors Derived from Lambda and Related Bacteriophages
 UNIT 1.9 Introduction to Lambda Phages
 UNIT 1.10 Lambda as a Cloning Vector
 UNIT 1.11 Plating Lambda Phage to Generate Plaques
 UNIT 1.12 Growing Lambda‐Derived Vectors
 UNIT 1.13 Preparing Lambda DNA from Phage Lysates
 Section IV Vectors Derived from Filamentous Phages
 UNIT 1.14 Introduction to Vectors Derived from Filamentous Phages
 UNIT 1.15 Preparing and Using M13‐Derived Vectors
 Section V Specialized Techniques
 UNIT 1.16 Recombineering: Genetic Engineering in Bacteria Using
Homologous Recombination
 UNIT 1.17 E. coli Genome Manipulation by P1 Transduction
 Chapter 2 Preparation and Analysis of DNA
 Introduction
 Section I Manipulation of DNA
 UNIT 2.1A Purification and Concentration of DNA from Aqueous Solutions
 UNIT 2.1B Purification of DNA by Anion‐Exchange Chromatography
 UNIT 2.2 Preparation of Genomic DNA from Mammalian Tissue
 UNIT 2.3 Preparation of Genomic DNA from Plant Tissue
 UNIT 2.4 Preparation of Genomic DNA from Bacteria
 Section II Resolution and Recovery of Large DNA Fragments
 UNIT 2.5A Agarose Gel Electrophoresis
 UNIT 2.5B Pulsed‐Field Gel Electrophoresis
 UNIT 2.6 Isolation and Purification of Large DNA Restriction Fragments from
Agarose Gels
 Section III Resolution and Recovery of Small DNA Fragments
 UNIT 2.7 Separation of Small DNA Fragments by Conventional Gel
Electrophoresis
 UNIT 2.8 Capillary Electrophoresis of DNA
 Section IV Analysis of DNA Sequences by Blotting and Hybridization
 UNIT 2.9A Southern Blotting
 UNIT 2.9B Dot and Slot Blotting of DNA
 UNIT 2.10 Hybridization Analysis of DNA Blots
 Section V Synthesis and Purification of Oligonucleotides
 UNIT 2.11 Synthesis and Purification of Oligonucleotides
 UNIT 2.12 Purification of Oligonucleotides Using Denaturing Polyacrylamide
Gel Electrophoresis
 Chapter 3 Enzymatic Manipulation of DNA and RNA
 Introduction
 Section I Restriction Endonucleases
 UNIT 3.1 Digestion of DNA with Restriction Endonucleases
 Section II Restriction Mapping
 UNIT 3.2 Mapping by Multiple Endonuclease Digestions
 UNIT 3.3 Mapping by Partial Endonuclease Digestions
 Section III Enzymes for Modifying and Radioactively Labeling Nucleic Acids
 UNIT 3.4 Reagents and Radioisotopes Used to Manipulate Nucleic Acids
 UNIT 3.5 DNA‐Dependent DNA Polymerases
 UNIT 3.6 Template‐Independent DNA Polymerases
 UNIT 3.7 RNA‐Dependent DNA Polymerases
 UNIT 3.8 RNA Polymerases
 UNIT 3.9 DNA Repair Enzymes
 UNIT 3.10 Phosphatases and Kinases
 UNIT 3.11 Exonucleases
 UNIT 3.12 Endonucleases
 UNIT 3.13 Ribonucleases
 UNIT 3.14 DNA Ligases
 UNIT 3.15 RNA Ligases
 Section IV Construction of Hybrid DNA Molecules
 UNIT 3.16 Subcloning of DNA Fragments
 UNIT 3.17 Constructing Recombinant DNA Molecules by PCR
 Section V Specialized Applications
 UNIT 3.18 Labeling and Colorimetric Detection of Nonisotopic Probes
 UNIT 3.19 Chemiluminescent Detection of Nonisotopic Probes
 UNIT 3.20 Recombinational Cloning
 UNIT 3.21 DNA Cloning and Engineering by Uracil Excision
 Chapter 4 Preparation and Analysis of RNA
 Introduction
 Section I Preparation of RNA from Eukaryotic and Prokaryotic Cells
 UNIT 4.1 Preparation of Cytoplasmic RNA from Tissue Culture Cells
 UNIT 4.2 Guanidine Methods for Total RNA Preparation
 UNIT 4.3 Phenol/SDS Method for Plant RNA Preparation
 UNIT 4.4 Preparation of Bacterial RNA
 UNIT 4.5 Preparation of Poly(A)+ RNA
 Section II Analysis of RNA Structure and Synthesis
 UNIT 4.6 S1 Analysis of Messenger RNA Using Single‐Stranded DNA Probes
 UNIT 4.7 Ribonuclease Protection Assay
 UNIT 4.8 Primer Extension
 UNIT 4.9 Analysis of RNA by Northern and Slot Blot Hybridization
 UNIT 4.10 Identification of Newly Transcribed RNA
 UNIT 4.11 RNA‐Seq: A Method for Comprehensive Transcriptome Analysis
 Chapter 5 Construction of Recombinant DNA Libraries
 Introduction
 Section I Overview of Recombinant DNA Libraries
 UNIT 5.1 Genomic DNA Libraries
 UNIT 5.2 cDNA Libraries
 Section II Preparation of Insert DNA from Genomic DNA
 UNIT 5.3 Size Fractionation Using Sucrose Gradients
 UNIT 5.4 Size Fractionation Using Agarose Gels
 Section III Preparation of Insert DNA from Messenger RNA
 UNIT 5.5 Conversion of mRNA into Double‐Stranded cDNA
 UNIT 5.6 Ligation of Linkers or Adapters to Double‐Stranded cDNA
 Section IV Production of Genomic DNA and cDNA Libraries
 UNIT 5.7 Production of a Genomic DNA Library
 UNIT 5.8A Production of a Complete cDNA Library
 UNIT 5.9 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
 Section V Amplification of Transformed or Packaged Libraries
 UNIT 5.10 Amplification of a Bacteriophage Library
 UNIT 5.11 Amplification of Cosmid and Plasmid Libraries
 UNIT 5.12 Normalizing cDNA Libraries
 Chapter 6 Screening of Recombinant DNA Libraries
 Introduction
 Section I Plating Libraries and Transfer to Filter Membranes
 UNIT 6.1 Plating and Transferring Bacteriophage Libraries
 UNIT 6.2 Plating and Transferring Cosmid and Plasmid Libraries
 Section II Hybridization with Radioactive Probes
 UNIT 6.3 Using DNA Fragments as Probes
 UNIT 6.4 Using Synthetic Oligonucleotides as Probes
 Section III Purification of Bacteriophage, Cosmid, and Plasmid Clones
 UNIT 6.5 Purification of Bacteriophage Clones
 UNIT 6.6 Purification of Cosmid and Plasmid Clones
 Section IV Screening with Antibodies
 UNIT 6.7 Immunoscreening of Fusion Proteins Produced in Lambda Plaques
 UNIT 6.8 Immunoscreening after Hybrid Selection and Translation
 Section V Yeast Artificial Chromosome Libraries
 UNIT 6.9 Overview of Strategies for Screening YAC Libraries and Analyzing
YAC Clones
 UNIT 6.10 Analysis of Isolated YAC Clones
 Section VI Specialized Strategies for Screening Libraries
 UNIT 6.11 Use of Monoclonal Antibodies for Expression Cloning
 UNIT 6.12 Recombination‐Based Assay (RBA) for Screening Bacteriophage
Lambda Libraries
 Chapter 7 DNA Sequencing
 Introduction
 UNIT 7.1 Overview of DNA Sequencing Strategies
 UNIT 7.2 Constructing Nested Deletions for Use in DNA Sequencing
 UNIT 7.3 Preparation of Templates for DNA Sequencing
 UNIT 7.4A DNA Sequencing by the Dideoxy Method
 UNIT 7.4B Dideoxy DNA Sequencing with Chemiluminescent Detection
 UNIT 7.5 DNA Sequencing by the Chemical Method
 UNIT 7.6 Denaturing Gel Electrophoresis for Sequencing
 UNIT 7.7 Computer Manipulation of DNA and Protein Sequences
 UNIT 7.8 Polony DNA Sequencing
 UNIT 7.9 Bisulfite Sequencing of DNA
 Chapter 8 Mutagenesis of Cloned DNA
 Introduction
 UNIT 8.1 Oligonucleotide‐Directed Mutagenesis without Phenotypic Selection
 UNIT 8.2A Mutagenesis with Degenerate Oligonucleotides: Creating Numerous
Mutations in a Small DNA Sequence
 UNIT 8.2B Gene Synthesis: Assembly of Target Sequences Using Mutually Priming
Long Oligonucleotides
 UNIT 8.3 Random Mutagenesis by PCR
 UNIT 8.4 Linker‐Scanning Mutagenesis of DNA
 UNIT 8.5 Directed Mutagenesis Using the Polymerase Chain Reaction
 Chapter 9 Introduction of DNA into Mammalian Cells
 Introduction
 Section I Transfection of DNA into Eukaryotic Cells
 UNIT 9.1 Calcium Phosphate Transfection
 UNIT 9.2 Transfection Using DEAE‐Dextran
 UNIT 9.3 Transfection by Electroporation
 UNIT 9.4 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid
Reagents
 UNIT 9.5 Selection of Transfected Mammalian Cells
 Section II Uses of Fusion Genes in Mammalian Transfection
 UNIT 9.6 Overview of Genetic Reporter Systems
 UNIT 9.7A Isotopic Assays for Reporter Gene Activity
 UNIT 9.7B Nonisotopic Assays for Reporter Gene Activity
 UNIT 9.7C Use of the A. Victoria Green Fluorescent Protein to Study Protein
Dynamics in Vivo
 UNIT 9.8 Direct Analysis of RNA after Transfection
 Section III Transduction of Genes Using Retrovirus Vectors
 UNIT 9.9 Overview of the Retrovirus Transduction System
 UNIT 9.10 Preparation of a Specific Retrovirus Producer Cell Line
 UNIT 9.11 Transient Transfection Methods for Preparation of High‐Titer
Retroviral Supernatants
 UNIT 9.12 Large‐Scale Preparation and Concentration of Retrovirus Stocks
 UNIT 9.13 Detection of Helper Virus in Retrovirus Stocks
 UNIT 9.14 Retrovirus Infection of Cells In Vitro and In Vivo
 Section IV Inactivation of Genes in Mammalian Cells
 UNIT 9.15 Human Somatic Cell Gene Targeting
 Chapter 10 Analysis of Proteins
 Introduction
 Section I Quantitation of Proteins
 UNIT 10.1A Spectrophotometric and Colorimetric Determination of Protein
Concentration
 UNIT 10.1B Quantitative Amino Acid Analysis
 Section II Electrophoretic Separation of Proteins
 UNIT 10.2A One‐Dimensional SDS Gel Electrophoresis of Proteins
 UNIT 10.2B One‐Dimensional Electrophoresis Using Nondenaturing Conditions
 UNIT 10.3 Two‐Dimensional Gel Electrophoresis Using the ISO‐DALT System
 UNIT 10.4 Two‐Dimensional Gel Electrophoresis
 UNIT 10.5 Overview of Digital Electrophoresis Analysis
 Section III Detection of Proteins
 UNIT 10.6 Staining Proteins in Gels
 UNIT 10.7 Detection of Proteins on Blot Transfer Membranes
 UNIT 10.8 Immunoblotting and Immunodetection
 Detection of Proteins
 Section IV Purification of Proteins by Conventional Chromatography
 UNIT 10.9 Gel‐Filtration Chromatography
 UNIT 10.10 Ion‐Exchange Chromatography
 UNIT 10.11A Immunoaffinity Chromatography
 UNIT 10.11B Metal‐Chelate Affinity Chromatography
 Section V Purification of Proteins by High‐Performance Liquid Chromatography
 UNIT 10.12 HPLC of Peptides and Proteins: Preparation and System Set‐Up
 UNIT 10.13 HPLC of Peptides and Proteins: Standard Operating Conditions
 UNIT 10.14 Reversed‐Phase Isolation of Peptides
 Section VI Specialized Applications
 UNIT 10.15 Purification of Recombinant Proteins and Study of Protein
Interaction by Epitope Tagging
 UNIT 10.16 Immunoprecipitation
 UNIT 10.17 Synthesizing Proteins In Vitro by Transcription and Translation of
Cloned Genes
 UNIT 10.18 Metabolic Labeling with Amino Acids
 UNIT 10.19 Isolation of Proteins for Microsequence Analysis
 UNIT 10.20 Capillary Electrophoresis of Proteins and Peptides
 UNIT 10.21 Overview of Peptide and Protein Analysis by Mass Spectrometry
 UNIT 10.22 Protein Identification and Characterization by Mass Spectrometry
 UNIT 10.23 Difference Gel Electrophoresis (DIGE) Using CyDye DIGE Fluor
Minimal Dyes
 UNIT 10.24 Solution Radioimmunoassay of Proteins and Peptides
 UNIT 10.25 Preparation of Proteins and Peptides for Mass Spectrometry
Analysis in a Bottom‐Up Proteomics Workflow
 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
 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
 Chapter 11 Immunology
 Introduction
 Section I Immunoassays
 UNIT 11.1 Conjugation of Enzymes to Antibodies
 UNIT 11.2 Enzyme‐Linked Immunosorbent Assays (ELISA)
 UNIT 11.3 Isotype Determination of Antibodies
 Section II Preparation of Monoclonal Antibodies
 UNIT 11.4 Immunization of Mice
 UNIT 11.5 Preparation of Myeloma Cells
 UNIT 11.6 Preparation of Mouse Feeder Cells for Fusion and Cloning
 UNIT 11.7 Fusion of Myeloma Cells with Immune Spleen Cells
 UNIT 11.8 Cloning of Hybridoma Cell Lines by Limiting Dilution
 UNIT 11.9 Freezing and Recovery of Hybridoma Cell Lines
 UNIT 11.10 Production of Monoclonal Antibody Supernatant and Ascites Fluid
 UNIT 11.11 Purification of Monoclonal Antibodies
 Section III Preparation of Polyclonal Antisera
 UNIT 11.12 Production of Polyclonal Antisera
 UNIT 11.13 In Vitro Antibody Production
 UNIT 11.14 Purification of Immunoglobulin G Fraction from Antiserum,
Ascites Fluid, or Hybridoma Supernatant
 Section IV Preparation of Antipeptide Antibodies
 UNIT 11.15 Introduction to Peptide Synthesis
 UNIT 11.16 Synthetic Peptides for Production of Antibodies that Recognize
Intact Proteins
 Section V Determination of Specific Antibody Titer and Isotype
 UNIT 11.17 Determination of the Specific Antibody Titer
 Section VI Preparation and Use of Specialized Antibodies
 UNIT 11.18 Identification of Polyol‐Responsive Monoclonal Antibodies for Use
in Immunoaffinity Chromatography
 Chapter 12 DNA‐Protein Interactions
 Introduction
 UNIT 12.1 Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
 UNIT 12.2 Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis
 UNIT 12.3 Methylation and Uracil Interference Assays for Analysis of Protein‐DNA
Interactions
 UNIT 12.4 DNase I Footprint Analysis of Protein‐DNA Binding
 UNIT 12.5 UV Crosslinking of Proteins to Nucleic Acids
 UNIT 12.6 Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity
Systems
 UNIT 12.7 Detection, Purification, and Characterization of cDNA Clones Encoding
DNA‐Binding Proteins
 UNIT 12.8 Rapid Separation of Protein‐Bound DNA from Free DNA Using Nitrocellulose
Filters
 UNIT 12.9 Analysis of DNA‐Protein Interactions Using Proteins Synthesized In Vitro
from Cloned Genes
 UNIT 12.10 Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity
Chromatography
 UNIT 12.11 Determination of Protein‐DNA Sequence Specificity by PCR‐Assisted
Binding‐Site Selection
 UNIT 12.12 Yeast One‐Hybrid Screening for DNA‐Protein Interactions
 Chapter 13 Yeast
 Introduction
 Section I Basic Techniques of Yeast Genetics
 UNIT 13.1 Preparation of Yeast Media
 UNIT 13.2 Growth and Manipulation of Yeast
 UNIT 13.3 Genome‐Wide Transposon Mutagenesis in Yeast
 UNIT 13.3B EMS and UV Mutagenesis in Yeast
 Section II Yeast Vectors
 UNIT 13.4 Yeast Cloning Vectors and Genes
 UNIT 13.6 Yeast Vectors and Assays for Expression of Cloned Genes
 Section III Manipulation of Yeast Genes
 UNIT 13.7 Introduction of DNA into Yeast Cells
 UNIT 13.8 Cloning Yeast Genes by Complementation
 UNIT 13.9 Manipulation of Plasmids from Yeast Cells
 UNIT 13.10 Manipulation of Cloned Yeast DNA
 Section IV Preparation of Yeast DNA, RNA, and Proteins
 UNIT 13.11 Preparation of Yeast DNA
 UNIT 13.12 Preparation of Yeast RNA
 UNIT 13.13 Preparation of Protein Extracts from Yeast
 Section V Schizosaccharomyces pombe
 UNIT 13.14 Overview of Schizosaccharomyces pombe
 UNIT 13.15 S. pombe Strain Maintenance and Media
 UNIT 13.16 Growth and Manipulation of S. pombe
 UNIT 13.17 Introduction of DNA into S. pombe Cells
 Chapter 14 In Situ Hybridization and Immunohistochemistry
 Introduction
 UNIT 14.1 Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
 UNIT 14.2 Cryosectioning
 UNIT 14.3 In Situ Hybridization to Cellular RNA
 UNIT 14.4 Detection of Hybridized Probe
 UNIT 14.5 Counterstaining and Mounting of Autoradiographed In Situ Hybridization
Slides
 UNIT 14.6 Immunohistochemistry
 UNIT 14.7 In Situ Hybridization and Detection Using Nonisotopic Probes
 UNIT 14.8 In Situ Polymerase Chain Reaction and Hybridization to Detect Low‐
Abundance Nucleic Acid Targets
 UNIT 14.9 Whole‐Mount In Situ Hybridization and Detection of RNAs in Vertebrate
Embryos and Isolated Organs
 UNIT 14.10 Principles and Application of Fluorescence Microscopy
 UNIT 14.11 Basic Confocal Microscopy
 UNIT 14.12 Measurement of In Situ Hybridization
 UNIT 14.13 Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis
 UNIT 14.14 Whole‐Mount Histochemical Detection of β‐Galactosidase Activity
 UNIT 14.15 Overview of Image Analysis, Image Importing, and Image Processing
using Freeware
 UNIT 14.16 Three‐Dimensional Reconstruction of Tissues
 UNIT 14.17 Using CellProfiler for Automatic Identification and Measurement of
Biological Objects in Images
 UNIT 14.18 Using Cell‐ID 1.4 with R for Microscope‐Based Cytometry
 UNIT 14.19 Visualization of Microscopy‐Based Spectral Imaging Data from Multi‐Label
Tissue Sections
 Chapter 15 The Polymerase Chain Reaction
 Introduction
 UNIT 15.1 Enzymatic Amplification of DNA by PCR: Standard Procedures and
Optimization
 UNIT 15.2 Direct DNA Sequencing of PCR Products
 UNIT 15.3 Ligation‐Mediated PCR for Genomic Sequencing and Footprinting
 UNIT 15.4 Molecular Cloning of PCR Products
 UNIT 15.5 Enzymatic Amplification of RNA by PCR (RT‐PCR)
 UNIT 15.6 cDNA Amplification Using One‐Sided (Anchored) PCR
 UNIT 15.7 Quantitation of Rare DNAs by PCR
 UNIT 15.8 High‐Throughput Real‐Time Quantitative Reverse Transcription PCR
 UNIT 15.9 Heat‐Activatable Primers for Hot‐Start PCR and Hot‐Start One‐Step RT‐PCR:
Endpoint and Real‐Time Experiments
 Chapter 16 Protein Expression
 Introduction
 Section I Expression of Proteins in Escherichia coli
 UNIT 16.1 Overview of Protein Expression in E. coli
 UNIT 16.2 Expression Using the T7 RNA Polymerase/Promoter System
 UNIT 16.3 Expression Using Vectors with Phage λ Regulatory Sequences
 UNIT 16.4A Introduction to Expression by Fusion Protein Vectors
 UNIT 16.4B Enzymatic and Chemical Cleavage of Fusion Proteins
 UNIT 16.5 Expression and Purification of lacZ and trpE Fusion Proteins
 UNIT 16.6 Expression and Purification of Maltose‐Binding Protein Fusions
 UNIT 16.7 Expression and Purification of Glutathione‐S‐Transferase Fusion
Proteins
 UNIT 16.8 Expression and Purification of Thioredoxin Fusion Proteins
 Section II Expression of Proteins in Insect Cells Using Baculovirus Vectors
 UNIT 16.9 Overview of the Baculovirus Expression System
 UNIT 16.10 Maintenance of Insect Cell Cultures and Generation of
Recombinant Baculoviruses
 UNIT 16.11 Expression and Purification of Recombinant Proteins Using the
Baculovirus System
 Section III Expression of Proteins in Mammalian Cells
 UNIT 16.12 Transient Expression of Proteins Using COS Cells
 UNIT 16.13 Expression and Purification of Epitope‐Tagged Multisubunit
Protein Complexes from Mammalian Cells
 UNIT 16.14 Inducible Gene Expression Using an Autoregulatory, Tetracycline‐
Controlled System
 UNIT 16.15 Overview of the Vaccinia Virus Expression System
 UNIT 16.16 Preparation of Cell Cultures and Vaccinia Virus Stocks
 UNIT 16.17 Generation of Recombinant Vaccinia Viruses
 UNIT 16.18 Characterization of Recombinant Vaccinia Viruses and Their
Products
 UNIT 16.19 Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase
Hybrid System
 UNIT 16.20 Expression of Proteins Using Semliki Forest Virus Vectors
 UNIT 16.21 Overview of the HIV‐1 Lentiviral Vector System
 UNIT 16.22 Generation of HIV‐1‐Based Lentiviral Vector Particles
 UNIT 16.23 Amplification Using CHO Cell Expression Vectors
 UNIT 16.24 Helper‐Dependent Adenoviral Vectors
 UNIT 16.25 Production of Recombinant Adeno‐Associated Viral Vectors for In
Vitro and In Vivo Use
 Chapter 17 Preparation and Analysis of Glycoconjugates
 Introduction
 Section I Special Considerations for Glycoproteins and Their Purification
 UNIT 17.1 Special Considerations for Glycoproteins and Their Purification
 UNIT 17.2 Special Considerations for Proteoglycans and Glycosaminoglycans
and Their Purification
 UNIT 17.3 Special Considerations for Glycolipids and Their Purification
 Section II Detection of Saccharides on Glycoconjugates
 UNIT 17.4 Metabolic Radiolabeling of Animal Cell Glycoconjugates
 UNIT 17.5 Chemical Labeling of Carbohydrates by Oxidation and Sodium
Borohydride Reduction
 UNIT 17.6 Detection and Analysis of Proteins Modified by O‐Linked N‐
Acetylglucosamine
 UNIT 17.7 Lectin Analysis of Proteins Blotted onto Filters
 UNIT 17.8 Detection of Glycophospholipid Anchors on Proteins
 UNIT 17.9 Direct Chemical Analysis of Glycoconjugates for Carbohydrates
 UNIT 17.10A Inhibition of N‐Linked Glycosylation
 UNIT 17.10B Inhibition of Glycolipid Biosynthesis
 UNIT 17.11 Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis
and Inhibitors of Glycoprotein and Proteoglycan Assembly
 Detection of Saccharides on Glycoconjugates
 Section III Release of Saccharides from Glycoconjugates
 UNIT 17.12 Sialidases
 UNIT 17.13A Endoglycosidase and Glycoamidase Release of N‐Linked Glycans
 UNIT 17.13B Analysis of Glycosaminoglycans with Polysaccharide Lyases
 UNIT 17.14A Preparation of Glycopeptides
 UNIT 17.14B Detection of Individual Glycosylation Sites on Glycoproteins
 UNIT 17.15A β‐Elimination for Release of O‐Linked Glycosaminoglycans from
Proteoglycans
 UNIT 17.15B β‐Elimination for Release of O‐GalNAc‐Linked Oligosaccharides
from Glycoproteins and Glycopeptides
 UNIT 17.16 Acid Hydrolysis for Release of Monosaccharides
 UNIT 17.17A Enzymatic Release of Oligosaccharides from Glycolipids
 UNIT 17.17B Endo‐β‐Galactosidases and Keratanase
 Section IV Analysis of Saccharides Released from Glycoconjugates
 UNIT 17.18 Analysis of Monosaccharides
 UNIT 17.19A Total Compositional Analysis by High‐Performance Liquid
Chromatography or Gas‐Liquid Chromatography
 UNIT 17.19B Composition of Labeled Monosaccharides from
Glycosaminoglycans
 UNIT 17.20 Analysis of Oligosaccharide Negative Charge by Anion‐Exchange
Chromatography
 UNIT 17.21A HPLC Methods for the Fractionation and Analysis of Negatively
Charged Oligosaccharides and Gangliosides
 UNIT 17.21B Fractionation and Analysis of Neutral Oligosaccharides by HPLC
 UNIT 17.22A Nitrous Acid Degradation of Glycosaminoglycans
 UNIT 17.22B Analysis of Disaccharides and Tetrasaccharides Released from
Glycosaminoglycans
 UNIT 17.23 Analysis of Sulfate Esters by Solvolysis or Hydrolysis
 Chapter 18 Analysis of Protein Phosphorylation
 Introduction
 UNIT 18.1 Overview of Protein Phosphorylation
 UNIT 18.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for
Immunoprecipitation
 UNIT 18.3 Phosphoamino Acid Analysis
 UNIT 18.4 Analysis of Phosphorylation of Unlabeled Proteins
 UNIT 18.5 Detection of Phosphorylation by Enzymatic Techniques
 UNIT 18.6 Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated
Peptides
 UNIT 18.7 Assays of Protein Kinases Using Exogenous Substrates
 UNIT 18.8 Permeabilization Strategies to Study Protein Phosphorylation
 UNIT 18.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites
 UNIT 18.10 Use of Protein Phosphatase Inhibitors
 UNIT 18.11 Design and Use of Analog‐Sensitive Protein Kinases
 UNIT 18.12 The Detection of MAPK Signaling
 UNIT 18.13 Isolation of Phosphopeptides by Immobilized Metal Ion Affinity
Chromatography
 UNIT 18.14 Analysis of Serine‐Threonine Kinase Specificity Using Arrayed Positional
Scanning Peptide Libraries
 UNIT 18.15 Visualization of Kinase Activity with FRET‐Based Activity Biosensors
 UNIT 18.16 Analysis of Protein Tyrosine Phosphatases and Substrates
 UNIT 18.17 Fluorescent Peptide Assays for Protein Kinases
 Chapter 19 Informatics for Molecular Biologists
 Introduction
 UNIT 19.1 Internet Basics for Biologists
 UNIT 19.2 Sequence Databases: Integrated Information Retrieval and Data
Submission
 UNIT 19.3 Sequence Similarity Searching Using the BLAST Family of Programs
 UNIT 19.4 Protein Databases on the Internet
 UNIT 19.5 Basic Protein Sequence Analysis
 UNIT 19.6 Analysis and Management of Microarray Gene Expression Data
 UNIT 19.7 Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries
 UNIT 19.8 Resources for Small Regulatory RNAs
 UNIT 19.9 The UCSC Genome Browser: What Every Molecular Biologist Should Know
 UNIT 19.10 Galaxy: A Web‐Based Genome Analysis Tool for Experimentalists
 Chapter 20 Analysis of Protein Interactions
 Introduction
 UNIT 20.1 Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
 UNIT 20.2 Affinity Purification of Proteins Binding to GST Fusion Proteins
 UNIT 20.3 Phage‐Based Expression Cloning to Identify Interacting Proteins
 UNIT 20.4 Surface Plasmon Resonance for Measurements of Biological Interest
 UNIT 20.5 Detection of Protein‐Protein Interactions by Coprecipitation
 UNIT 20.6 Identification of Protein Interactions by Far Western Analysis
 UNIT 20.7 Two‐Hybrid Dual Bait System
 UNIT 20.8 Interaction Trap/Two‐Hybrid System to Identify Loss‐of‐Interaction Mutant
Proteins
 Chapter 21 Chromatin Assembly and Analysis
 Introduction
 UNIT 21.1 Micrococcal Nuclease Analysis of Chromatin Structure
 UNIT 21.2 Separation of Histone Variants and Post‐Translationally Modified Isoforms
by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis
 UNIT 21.3 Chromatin Immunoprecipitation for Determining the Association of
Proteins with Specific Genomic Sequences In Vivo
 UNIT 21.4 DNase I and Hydroxyl Radical Characterization of Chromatin Complexes
 UNIT 21.5 Isolation of Histones and Nucleosome Cores from Mammalian Cells
 UNIT 21.6 Assembly of Nucleosomal Templates by Salt Dialysis
 UNIT 21.7 Chromatin Assembly Using Drosophila Systems
 UNIT 21.8 Analysis of Protein Co‐Occupancy by Quantitative Sequential Chromatin
Immunoprecipitation
 UNIT 21.9 Defining In Vivo Targets of Nuclear Proteins by Chromatin
Immunoprecipitation and Microarray Analysis
 UNIT 21.10 Identifying Chromosomal Targets of DNA‐Binding Proteins by Sequence
Tag Analysis of Genomic Enrichment (STAGE)
 UNIT 21.11 Mapping Chromatin Interactions by Chromosome Conformation Capture
 UNIT 21.12 Paired‐End diTagging for Transcriptome and Genome Analysis
 UNIT 21.13 ChIP‐chip for Genome‐Wide Analysis of Protein Binding in Mammalian
Cells
 UNIT 21.14 Chromosome Conformation Capture Carbon Copy Technology
 UNIT 21.15 Chromatin Interaction Analysis Using Paired‐End Tag Sequencing
 UNIT 21.16 Mapping Networks of Protein‐Mediated Physical Interactions Between
Chromatin Elements
 UNIT 21.17 Methylation‐Sensitive Single‐Molecule Analysis of Chromatin Structure
 UNIT 21.18 Installation of Site‐Specific Methylation into Histones Using Methyl Lysine
Analogs
 UNIT 21.19 ChIP‐Seq: A Method for Global Identification of Regulatory Elements in
the Genome
 Chapter 22 Nucleic Acid Arrays
 Introduction
 UNIT 22.1 Overview of Nucleic Acid Arrays
 UNIT 22.2 Preparation of mRNA for Expression Monitoring
 UNIT 22.3 Profiling Human Gene Expression with cDNA Microarrays
 UNIT 22.4 Overview of mRNA Expression Profiling Using DNA Microarrays
 UNIT 22.5 Pattern Discovery in Expression Profiling Data
 Chapter 23 Manipulating the Mouse Genome
 Introduction
 UNIT 23.1 Overview of Gene Targeting by Homologous Recombination
 UNIT 23.2 Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation
 UNIT 23.3 Mouse Embryonic Stem (ES) Cell Culture
 UNIT 23.4 Mouse Embryonic Stem (ES) Cell Isolation
 UNIT 23.5 Production of a Heterozygous Mutant Cell Line by Homologous
Recombination (Single Knockout)
 UNIT 23.6 Production of a Homozygous Mutant Embryonic Stem Cell Line (Double
Knockout)
 UNIT 23.7 Chimeric Mouse Production by Microinjection
 UNIT 23.8 Mouse Colony Management
 UNIT 23.9 Transgenic Mouse Production By Zygote Injection
 UNIT 23.10 Transgenic Mouse Colony Management
 UNIT 23.11 Modification and Production of BAC Transgenes
 UNIT 23.12 Regulation of Transgene Expression Using Tetracycline
 UNIT 23.13 Recombineering‐Based Procedure for Creating Cre/loxP Conditional
Knockouts in the Mouse
 Chapter 24 Generation and Use of Combinatorial Libraries
 UNIT 24.1 Overview of Receptors from Combinatorial Nucleic Acid and Protein
Libraries
 UNIT 24.2 Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection
 UNIT 24.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter
Immobilization
 UNIT 24.4 Peptide Aptamers: Dominant “Genetic” Agents for Forward and Reverse
Analysis of Cellular Processes
 UNIT 24.5 Protein Selection Using mRNA Display
 UNIT 24.6 Directed Evolution of Proteins In Vitro Using Compartmentalization in
Emulsions
 Chapter 25 Discovery and Analysis of Differentially Expressed Genes in Single Cells and Cell
Populations
 Introduction
 Section A Nucleic Acid Amplification from Individual Cells
 Section B Molecular Methods for Discovery of Differentially Expressed Genes
 Chapter 26 Gene Silencing
 Introduction
 UNIT 26.1 Overview of RNA Interference and Related Processes
 UNIT 26.2 Gene Silencing by RNAi in Mammalian Cells
 UNIT 26.3 RNA Interference in Caenorhabditis Elegans
 UNIT 26.4 Cloning of Small RNA Molecules
 UNIT 26.5 RNA Interference in Cultured Drosophila Cells
 UNIT 26.6 RNAi in Transgenic Plants
 UNIT 26.7 Analysis of Small Endogenous RNAs
 UNIT 26.8 Using Morpholinos to Control Gene Expression
 Chapter 27 RNA‐Protein Interactions
 Introduction
 UNIT 27.1 Agarose Gel Separation/Isolation of RNA‐Protein Complexes
 UNIT 27.2 Identification of RNA Binding Proteins by UV Cross‐Linking
 UNIT 27.3 Purification of Functional RNA‐Protein Complexes using MS2‐MBP
 UNIT 27.4 RNA Immunoprecipitation for Determining RNA-Protein Associations In
Vivo
 Chapter 28 Mammalian Cell Culture
 Introduction
 UNIT 28.1 Preparation, Culture, and Immortalization of Mouse Embryonic Fibroblasts
 UNIT 28.2 Isolation and Immortalization of Lymphocytes
 UNIT 28.3 Establishment and Culture of Human Skin Fibroblasts
 Chapter 29 Mouse Phenotyping
 Introduction
 Section A General Considerations in Mouse Phenotyping
 UNIT 29A.1 Uses of Forward and Reverse Genetics in Mice to Study Gene
Function
 UNIT 29A.2 Minimizing Variation Due to Genotype and Environment
 UNIT 29A.3 Collection of Blood and Plasma from the Mouse
 UNIT 29A.4 Tissue Collection for Systematic Phenotyping in the Mouse
 Section B Metabolic Exploration of the Mouse
 UNIT 29B.1 Evaluation of Energy Homeostasis
 UNIT 29B.2 Lipid and Bile Acid Analysis
 UNIT 29B.3 Evaluation of Glucose Homeostasis
 UNIT 29B.4 Histopathology in Mouse Metabolic Investigations
 UNIT 29B.5 Dietary Manipulation of Mouse Metabolism
 Chapter 30 Metabolomics
 Introduction
 UNIT 30.1 Untargeted Metabolomics
 Appendix 1 Standard Measurements, Data, and Abbreviations
 APPENDIX 1B Useful Measurements and Data
 APPENDIX 1C Characteristics of Amino Acids
 APPENDIX 1D Characteristics of Nucleic Acids
 APPENDIX 1E Radioactivity
 APPENDIX 1F Safe Use of Radioisotopes
 APPENDIX 1G Centrifuges and Rotors
 APPENDIX 1H Safe Use of Hazardous Chemicals
 APPENDIX 1I Commonly Used Detergents
 APPENDIX 1J Common Conversion Factors
 APPENDIX 1K Compendium of Drugs Commonly Used in Molecular Biology Research
 Appendix 2 Commonly Used Reagents and Equipment
 Appendix 3 Commonly Used Techniques in Biochemistry and Molecular Biology
 APPENDIX 3A Detection and Quantitation of Radiolabeled Proteins and DNA in Gels
and Blots
 APPENDIX 3B Silanizing Glassware
 APPENDIX 3C Dialysis and Ultrafiltration
 APPENDIX 3D Quantitation of DNA and RNA with Absorption and Fluorescence
Spectroscopy
 APPENDIX 3E Introduction of Restriction Enzyme Recognition Sequences by Silent
Mutation
 APPENDIX 3F Techniques for Mammalian Cell Tissue Culture
 APPENDIX 3G Importing Biological Materials
 APPENDIX 3H Kinetic Assay Methods
 APPENDIX 3I Statistics for the Molecular Biologist: Group Comparisons
 Appendix 4 Suppliers
 Appendix 5 Vectors
 Appendix 6 Pathway Modulators and Inhibitors
 APPENDIX 6 Pathway Modulators and Inhibitors