9780471329381

Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology

by ; ; ; ;
  • ISBN13:

    9780471329381

  • ISBN10:

    047132938X

  • Edition: 4th
  • Format: Paperback
  • Copyright: 1999-04-01
  • Publisher: John Wiley & Sons Inc
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Summary

Expanded to 2 volumes, "Short Protocols in Molecular" "Biology, Fifth Edition," provides condensed descriptions of more than 700 methods compiled from Current Protocols in Molecular Biology. Includes new chapters on chromatin assembly and analysis, nucleic acid arrays, generation and use of combinatorial libraries, discovery and analysis of differentially expressed genes in single cells and cell populations. The book is specifically designed to provide quick access to step-by-step instructions for the essential methods used in every major area of molecular biological research "Short Protocols in Molecular Biology, Fifth Edition" is an authoritative and indispensable guide for all life scientists, researchers, and students at the graduate and advanced undergraduate level.

Table of Contents

Preface xxiii
Contributors xxvii
Escherichia Coli, Plasmids, and Bacteriophages
Introduction
1-1(1)
Media Preparation and Bacteriological Tools
1-2(1)
Minimal Media
1-2(1)
Rich Media
1-3(1)
Solid Media
1-3(1)
Top Agar
1-4(1)
Stab Agar
1-4(1)
Tools
1-4(1)
Growth in Liquid Media
1-5(1)
Basic Protocol 1: Growing an Overnight Culture
1-5(1)
Basic Protocol 2: Growing Larger Cultures
1-5(1)
Basic Protocol 3: Monitoring Growth
1-6(1)
Growth on Solid Media
1-6(1)
Basic Protocol 1: Titering and Isolating Bacterial Colonies by Serial Dilutions
1-6(1)
Basic Protocol 2: Isolating Single Colonies by Streaking a Plate
1-7(1)
Basic Protocol 3: Isolating Single Colonies by Spreading a Plate
1-7(1)
Support Protocol 1: Replica Plating
1-8(1)
Support Protocol 2: Strain Storage and Revival
1-8(1)
Selected Topics from Classical Bacterial Genetics
1-9(1)
Maps of Plasmids
1-13(1)
Choosing a Plasmid Vector
1-15(1)
Minipreps of Plasmid DNA
1-22(1)
Basic Protocol 1: Alkaline Lysis Miniprep
1-22(1)
Alternate Protocol: Alkaline Lysis in 96-Well Microtiter Dishes
1-22(1)
Basic Protocol 2: Boiling Miniprep
1-23(1)
Support Protocol: Storage of Plasmid DNA
1-24(1)
Large-Scale Preparation of Plasmid DNA
1-24(1)
Preparation of Crude Lysates
1-24(1)
Basic Protocol 1: Alkaline Lysis
1-24(1)
Basic Protocol 2: CsCl/Ethidium Bromide Equilibrium Centrifugation
1-25(1)
Alternate Protocol: Plasmid DNA Purification by Anion-Exchange or Size-Exclusion Chromatography
1-26(1)
Introduction of Plasmid DNA into Cells
1-27(1)
Basic Protocol 1: Transformation Using Calcium Chloride
1-27(1)
Alternate Protocol: One-Step Preparation and Transformation of Competent Cells
1-28(1)
Basic Protocol 2: High-Efficiency Transformation by Electroporation
1-29(1)
Introduction to Lambda Phages
1-30(1)
Lytic Growth
1-31(1)
Lysogenic Growth
1-32(1)
Lambada as a Cloning Vector
1-33(1)
Advantages of Using Lambada
1-33(1)
Selections for Inserted DNA
1-33(1)
Maps of Lambda-Derived Cloning Vectors
1-36(1)
Plating Lambda Phage to Generate Plaques
1-36(1)
Basic Protocol 1: Isolating a Single Plaque by Titering Serial Dilutions
1-36(1)
Basic Protocol 2: Phage Transfection and In Vitro Packaging
1-37(1)
Growing Lambada-Derived Vectors
1-38(1)
Basic Protocol: Making a Stock of Phage by Plate Lysis
1-38(1)
Alternate Protocol: Making a Liquid Lysate
1-38(1)
Preparing Lambda DNA from Phage Lysates
1-39(1)
Basic Protocol: Preparing DNA by Step- and Equilibrium-Gradient Centrifugation
1-39(1)
Introduction to Vectors Derived from Filamentous Phages
1-41(1)
Preparing and Using M13-Derived Vectors
1-44(1)
Basic Protocol: Preparing Single-Stranded DNA from Plasmids Using Helper Phage
1-44(1)
Preparation and Analysis of DNA
Introduction
2-1(1)
Purification and Concentration of DNA from Aqueous Solutions
2-3(1)
Basic Protocol: Phenol Extraction and Ethanol Precipitation of DNA
2-3(1)
Alternate Protocol 1: Precipitation of DNA Using Isopropanol
2-4(1)
Support Protocol 1: Buffering Phenol and Preparing Phenol/Chloroform/Isoamyl Alchohol
2-4(1)
Support Protocol 2: Concentration of DNA Using Butanol
2-5(1)
Support Protocol 3: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction
2-5(1)
Alternate Protocol 2: Purification of DNA Using Glass Beads
2-6(1)
Alternate Protocol 3: Purification and Concentration of RNA and Dilute Solutions of DNA
2-6(1)
Alternate Protocol 4: Removal of Low-Molecular-Weight Oligonucleotides and Triphosphates by Ethanol Precipitation
2-7(1)
Purification of DNA by Anion-Exchange Chromatography
2-8(1)
Preparation of Genomic DNA from Mammalian Tissue
2-9(1)
Preparation of Genomic DNA from Plant Tissue
2-10(1)
Basic Protocol: Preparation of Plant DNA Using CsCl Centrifugation
2-10(1)
Alternate Protocol: Preparation of Plant DNA Using CTAB
2-11(1)
Preparation of Genomic DNA from Bacteria
2-12(1)
Basic Protocol: Miniprep of Bacterial Genomic DNA
2-12(1)
Alternate Protocol: Large-Scale CsCl Prep of Bacterial Genomic DNA
2-13(1)
Support Protocol: Removal of Polysaccharides from Existing Genomic DNA Preps
2-14(1)
Agarose Gel Electrophoresis
2-14(1)
Basic Protocol: Resolution of Large DNA Fragments on Agarose Gels
2-14(1)
Support Protocol: Minigels and Midigels
2-16(1)
Pulsed-Field Gel Electrophoresis
2-16(1)
Basic Protocol: Field-Inversion Electrophoresis
2-16(1)
Alternate Protocol: Chef Electrophoresis
2-17(1)
Support Protocol: Preparation of High-Molecular-Weight DNA Samples and Size Markers
2-18(1)
Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
2-20(1)
Basic Protocol 1: Electroelution from Agarose Gels
2-20(1)
Basic Protocol 2: Electrophoresis onto NA-45 Paper
2-21(1)
Basic Protocol 3: Isolation of DNA Fragments Using Low Gelling/Melting Temperature Agarose Gels
2-22(1)
Alternate Protocol 1: Recovery of DNA from Low Gelling/Melting Temperature Agarose Gels Using β-Agarase Digestion
2-23(1)
Alternate Protocol 2: Recovery of DNA from Low Gelling/Melting Temperature Agarose Using Glass Beads
2-23(1)
Support Protocol: Rapid Estimation of DNA Concentration by Ethidium Bromide Dot Quantitation
2-24(1)
Separation of Small DNA Fragments by Conventional Gel Electrophoresis
2-24(1)
Basic Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis
2-24(1)
Alternate Protocol: Electroelution of Small DNA Fragments from Polyacrylamide Gels
2-27(1)
Basic Protocol 2: Sieving Agarose Gel Electrophoresis
2-27(1)
Capillary Electrophoresis of DNA
2-28(1)
Instrumentation
2-28(1)
Separation Theory
2-29(1)
Strategic Planning
2-29(1)
Basic Protocol 1: Separation of Oligonucleotides
2-31(1)
Basic Protocol 2: Quantitative PCR Analysis
2-33(1)
Alternate Protocol: Genotyping
2-34(1)
Southern Blotting
2-34(1)
Basic Protocol: Southern Blotting onto a Nylon or Nitrocellulose Membrane with High-Salt Buffer
2-34(1)
Support Protocol: Calibration of a UV Transilluminator
2-37(1)
Alternate Protocol 1: Southern Blotting onto a Nylon Membrane with an Alkaline Buffer
2-37(1)
Alternate Protocol 2: Southern Blotting by Downward Capillary Transfer
2-38(1)
Alternate Protocol 3: Electroblotting from a Polyacrylamide Gel to a Nylon Membrane
2-38(1)
Dot and Slot Blotting of DNA
2-39(1)
Basic Protocol: Dot and Slot Blotting of DNA onto Uncharged Nylon and Nitrocellulose Membranes Using a Manifold
2-40(1)
Alternate Protocol 1: Dot and Slot Blotting of DNA onto a Positively Charged Nylon Membrane Using a Manifold
2-41(1)
Alternate Protocol 2: Manual Preparation of a DNA Dot Blot
2-41(1)
Hybridization Analysis of DNA Blots
2-42(1)
Basic Protocol: Hybridization Analysis of a DNA Blot with a Radiolabeled DNA Probe
2-42(1)
Alternate Protocol: Hybridization Analysis of a DNA Blot with a Radiolabeled RNA Probe
2-44(1)
Support Protocol: Removal of Probes from Hybridized Membranes
2-48(1)
Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis
2-49(1)
Enzymatic Manipulation of DNA and RNA
Introduction
3-1(1)
Digestion of DNA with Restriction Endonucleases
3-2(1)
Basic Protocol: Digesting a Single DNA Sample with a Single Restriction Endonuclease
3-2(1)
Alternate Protocol 1: Digesting DNA with Multiple Restriction Endonucleases
3-3(1)
Alternate Protocol 2: Digesting Multiple Samples of DNA
3-6(1)
Alternate Protocol 3: Partial Digestion of DNA with Restriction Endonucleases
3-7(1)
Support Protocol: Methylation of DNA
3-7(1)
Mapping by Multiple Endonuclease Digestions
3-8(1)
Mapping by Partial Endonuclease Digestions
3-9(1)
Reagents and Radioisotopes Used to Manipulate Nucleic Acids
3-9(1)
Stock Solutions
3-9(1)
10x Enzyme Buffers
3-10(1)
Enzyme Reaction Conditions and Applications
3-13(1)
Nucleoside Triphosphates
3-13(1)
Radioisotopes for Labeling Nucleic Acids
3-16(1)
Basic Protocol: Measuring Radioactivity in DNA and RNA by Acid Precipitation
3-16(1)
Alternate Protocol: Spin-Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors
3-17(1)
DNA-Dependent DNA Polymerases
3-18(1)
Klenow Fragment of E. coli DNA Polymerase I
3-18(1)
Basic Protocol 1: Labeling the 3' Ends of DNA
3-19(1)
Basic Protocol 2: Repairing 3' or 5' Overhanging Ends to Generat Blunt Ends
3-20(1)
Basic Protocol 3: Labeling of DNA by Random Oligonucleotide-Primed Synthesis
3-20(1)
T4 DNA Polymerase
3-21(1)
Native T7 DNA Polymerase
3-23(1)
Modified T7 DNA Polymerase
3-23(1)
Taq DNA Polymerase
3-24(1)
Template-Independent DNA Polymerases
3-25(1)
RNA-Dependent DNA Polymerases
3-26(1)
DNA-Dependent RNA Polymerases
3-27(1)
DNA-Independent RNA Polymerases
3-28(1)
Phosphatases and Kinases
3-28(1)
Alkaline Phosphatases: BAP and CIP
3-28(1)
T4 Polynucleotide Kinase
3-29(1)
Basic Protocol 1: Labeling 5' Ends by the Forward Reaction
3-29(1)
Basic Protocol 2: Labeling 5' Termini by the Exchange Reaction
3-29(1)
Exonucleases
3-30(1)
Single-Stranded 5'→3' and 3'→5' Exonucleases
3-30(1)
Double-Stranded 5'→3' Exonucleases
3-30(1)
Double-Stranded 3'→5' Exonucleases
3-30(1)
Endonucleases
3-31(1)
Bal 31 Nuclease
3-31(1)
S1 Nuclease
3-32(1)
Mung Bean Nuclease
3-32(1)
Micrococcal Nuclease
3-33(1)
Deoxyribonuclease I (DNase I)
3-33(1)
Ribonucleases
3-34(1)
Ribonuclease A
3-34(1)
Ribonuclease H
3-34(1)
Ribonuclease T1
3-35(1)
DNA Ligases
3-36(1)
T4 DNA Ligase
3-36(1)
E. coli DNA Ligase
3-36(1)
RNA Ligases
3-36(1)
T4 RNA Ligase
3-36(1)
Subcloning of DNA Fragments
3-37(1)
Basic Protocol
3-37(1)
Alternate Protocol: Ligation of DNA Fragments in Gel Slices
3-39(1)
Constructing Recombinant DNA Molecules by the Polymerase Chain Reaction
3-39(1)
Basic Protocol: Subcloning DNA Fragments
3-39(1)
Labeling and Colorimetric Detection of Nonisotopic Probes
3-43(1)
Basic Protocol 1: Preparation of Biotinylated Probes by Nick Translation
3-43(1)
Basic Protocol 2: Preparation of Biotinylated Probes by Random Oligonucleotide-Primed Synthesis
3-45(1)
Support Protocol: Colorimetric Detection of Biotinylated Probes
3-45(1)
Alternate Protocol: Preparation and Detection of Digoxigenin-Labeled DNA Probes
3-47(1)
Chemiluminescent Detection of Nonisotopic Probes
3-47(1)
Basic Protocol: Chemiluminescent Detection of Biotinylated Probes
3-47(1)
Alternate Protocol: Chemiluminescent Detection of Digoxigenin-Labeled Probes
3-50(1)
Support Protocol: Calibrating an Ultraviolet Light Source
3-50(1)
Preparation and Analysis of RNA
Introduction
4-1(1)
Preparation of Cytoplasmic RNA from Tissue Culture Cells
4-2(1)
Basic Protocol
4-2(1)
Support Protocol: Removal of Contaminating DNA
4-3(1)
Guanidinium Methods for Total RNA Preparation
4-4(1)
Basic Protocol: Single-Step RNA Isolation from Cultured Cells or Tissues
4-4(1)
Alternate Protocol 1: CsCl Purification of RNA from Cultured Cells
4-5(1)
Alternate Protocol 2: CsCl Purification of RNA from Tissue
4-6(1)
Phenol/SDS Method for Plant RNA Preparation
4-7(1)
Preparation of Bacterial RNA
4-8(1)
Basic Protocol 1: Isolation of High-Quality RNA from Gram-Negative Bacteria
4-8(1)
Basic Protocol 2: Isolation of RNA from Gram-Positive Bacteria
4-10(1)
Alternate Protocol: Rapid Isolation of RNA from Gram-Negative Bacteria
4-11(1)
Preparation of Poly(A)+RNA
4-11(1)
S1 Analysis of Messenger RNA Using Single-Stranded DNA Probes
4-12(1)
Basic Protocol: S1 Analysis of mRNA Using M13 Template
4-12(1)
Alternate Protocol 1: Synthesis of Single-Stranded Probe from Double-Stranded Plasmid Template
4-15(1)
Alternate Protocol 2: Quantitative S1 Analysis of mRNA Using Oligonucleotide Probes
4-15(1)
Support Protocol: Controls for Quantitative S1 Analysis of mRNA
4-16(1)
Ribonuclease Protection Assay
4-17(1)
Basic Protocol
4-17(1)
Support Protocol 1: Gel Purification of RNA Probes
4-18(1)
Support Protocol 2: Preparation of Template DNA
4-19(1)
Primer Extension
4-19(1)
Analysis of RNA by Northern and Slot Blot Hybridization
4-21(1)
Basic Protocol: Northern Hybridization of RNA Fractionated by Agarose-Formaldehyde Gel Electrophoresis
4-22(1)
Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment
4-24(1)
Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting
4-25(1)
Support Protocol: Removal of Probes from Northern Blots
4-26(1)
Construction of Recombinant DNA Libraries
Introduction
5-1(1)
Overview of Genomic DNA Libraries
5-2(1)
Representation and Randomness
5-3(1)
Subgenomic Libraries
5-3(1)
Vectors for Genomic DNA Libraries
5-3(1)
Overview of cDNA Libraries
5-4(1)
Amplification of a Bacteriophage Library
5-5(1)
Amplification of Cosmid and Plasmid Libraries
5-6(1)
Screening of Recombinant DNA Libraries
Introduction
6-1(1)
Plating and Transferring Bacteriophage Libraries
6-3(1)
Plating and Transferring Cosmid and Plasmid Libraries
6-4(1)
Using DNA Fragments as Probes
6-6(1)
Basic Protocol: Hybridization in Formamide
6-6(1)
Alternate Protocol: Hybridization in Aqueous Solution
6-6(1)
Using Synthetic Oligonucleotides as Probes
6-7(1)
Basic Protocol 1: Hybridization in Sodium Chloride/Sodium Citrate (SSC)
6-7(1)
Basic Protocol 2: Hybridization in Tetramethylammonium Chloride (TMAC)
6-8(1)
Support Protocol: Labeling the 5' Ends of Mixed Oligonucleotides
6-10(1)
Purification of Bacteriophage Clones
6-11(1)
Purification of Cosmid and Plasmid Clones
6-11(1)
Immunoscreening of Fusion Proteins Produced in Lambda Plaques
6-12(1)
Basic Protocol: Screening a λgt11 Expression Library with Antibodies
6-12(1)
Alternate Protocol: Induction of Fusion Protein Expression with IPTG Prior to Screening with Antibodies
6-13(1)
Immunoscreening after Hybrid Selection and Translation
6-14(1)
Overview of Strategies for Screening YAC Libraries and Analyzing YAC Clones
6-16(1)
Generating YAC Libraries
6-16(1)
YAC Library Screening by a Core Laboratory
6-16(1)
Designing a Locus-Specific PCR Assay for Screening
6-18(1)
Analyzing Individual YAC Clones
6-18(1)
Construction and Analysis of a YAC-Insert Sublibrary
6-19(1)
Analysis of Isolated YAC Clones
6-20(1)
Basic Protocol 1: Propagation and Storage of YAC-Containing Yeast Strains
6-20(1)
Basic Protocol 2: Preparation of YAC-Containing DNA from Yeast Clones for Analysis by Southern Blotting
6-20(1)
Basic Protocol 3: Preparation of Yeast Chromosomes in Agarose Plugs for Pulsed- Field Gel Electrophoresis
6-22(1)
Basic Protocol 4: End-Fragment Analysis Using PCR Amplification
6-23(1)
Alternate Protocol: End-Fragment Analysis by Subcloning into a Bacterial Plasmid Vector
6-26(1)
Support Protocol: Design and Preparation of pUC19-ES and pUC19-HS Subcloning Vector
6-28(1)
Basic Protocol 5: Preparation of High-Molecular-Weight YAC-Containing Yeast DNA in Solution
6-28(1)
Basic Protocol 6: Preparation and Analysis of a YAC-Insert Sublibrary
6-30(1)
DNA Sequencing
Overview of DNA Sequencing Methods
7-1(1)
DNA Sequencing Strategies
7-8(1)
Dideoxy Sequencing
7-8(1)
Chemical Sequencing
7-9(1)
Constructing Nested Deletions for Use in DNA Sequencing
7-11(1)
Basic Protocol 1: Using Exonuclease III to Construct Unidirectional Deletions
7-11(1)
Support Protocol 1: Protection of DNA from Exonuclease III Digestion Using [α35S]dNTPs
7-15(1)
Basic Protocol 2: Using Bal 31 Nuclease to Construct Nested Deletions
7-15(1)
Support Protocol 2: Preparation of M13mp Sequencing Vector DNA for Subcloning of Bal 31-Digested DNA Fragments
7-20(1)
Preparation of Templates for DNA Sequencing
7-21(1)
Basic Protocol 1: Preparation of Single-Stranded M13 Phage DNA
7-21(1)
Basic Protocol 2: Preparation of λDNA from Small-Scale Lysates
7-22(1)
Basic Protocol 3: Miniprep of Double-Stranded Plasmid DNA for Dideoxy Sequencing
7-23(1)
Basic Protocol 4: Alkali Denaturation of Double-Stranded Plasmid DNA for Dideoxy Sequencing
7-24(1)
Basic Protocol 5: Preparation of Plasmid DNA from an E. coli Colony or Phage DNA from a Plaque for Thermal Cycle Sequencing
7-25(1)
DNA Sequencing by the Dideoxy Method
7-25(1)
Basic Protocol 1: Labeling/Termination Sequencing Reactions Using Sequenase (Modified T7 DNA Polymerase)
7-26(1)
Alternate Protocol 1: Using Mn2+ in the Labeling/Termination Reactions
7-30(1)
Alternate Protocol 2: Using Taq DNA Polymerase in the Sanger Procedure
7-30(1)
Alternate Protocol 3: One-Step Sequencing Reactions Using 5'-End-Labeled Primers
7-31(1)
Basic Protocol 2: Thermal Cycle Sequencing Reactions Using αLabeled Nucleotides
7-32(1)
Alternate Protocol 4: Thermal Cycle Sequencing Reactions Using 5'-End-Labeled Primers
7-33(1)
Alternate Protocol 5: Cycle Sequencing Using Fluorescence Dye-Labeled Primer (Dyeprimer) or Terminator (Dyeterminator)
7-35(1)
Dideoxy DNA Sequencing with Chemiluminescent Detection
7-37(1)
Basic Protocol: DNA Sequencing Using Biotinylated Primers with Chemiluminescent Detection
7-38(1)
Alternate Protocol 1: Two-Step (Indirect) Detection Using Streptavidin and Biotinylated Alkaline Phosphatase
7-40(1)
Alternate Protocol 2: Sequencing with Hapten-Labeled Primers and Detection with Antibody-Alkaline Phosphatase Conjugates
7-41(1)
Denaturing Gel Electrophoresis for Sequencing
7-42(1)
Basic Protocol: Pouring, Running, and Processing Sequencing Gels
7-43(1)
Alternate Protocol 1: Buffer-Gradient Sequencing Gels
7-46(1)
Alternate Protocol 2: Electrolyte-Gradient Sequencing Gels
7-46(1)
Alternate Protocol 3: Formamide-Containing Sequencing Gels
7-47(1)
Computer Manipulation of DNA and Protein Sequences
7-47(1)
Sequence Data Entry
7-48(1)
Sequence Data Verification and Assembly
7-50(1)
Restriction Mapping
7-52(1)
Prediction of Nucleic Acid Structure
7-54(1)
Oligonucleotide Design Strategy
7-55(1)
Identification of Protein-Coding Regions
7-57(1)
Homology Searching
7-58(1)
Genetic Sequence Databases and Other Electronic Resources Available to Molecular Biologists
7-60(1)
Appendix
7-60(1)
Mutagenesis of Cloned DNA
Introduction
8-1(1)
Oligonucleotide-Directed Mutagenesis Without Phenotypic Selection
8-2(1)
Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence
8-5(1)
Gene Synthesis: Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides
8-8(1)
Region-Specific Mutagenesis
8-9(1)
Basic Protocol
8-9(1)
Support Protocol: Enrichment of Mutant Clones
8-12(1)
Linker-Scanning Mutagenesis of DNA
8-12(1)
Basic Protocol: Linker Scanning Using Nested Deletions and Complementary Oligonucleotides
8-12(1)
Alternate Protocol: Linker Scanning Using Oligonucleotide-Directed Mutagenesis
8-14(1)
Site-Directed Mutagenesis by the Polymerase Chain Reaction
8-15(1)
Basic Protocol 1: Introduction of Restriction Endonuclease Sites by PCR
8-15(1)
Basic Protocol 2: Introduction of Point Mutations by PCR
8-16(1)
Alternate Protocol: Introduction of a Point Mutation by Sequential PCR Steps
8-21(1)
Introduction of DNA into Mammalian Cells
Overview of Transfection Methods
9-1(1)
Calcium Phosphate Transfection
9-6(1)
Basic Protocol: Transfection Using Calcium Phosphate-DNA Precipitate Formed in HEPES
9-6(1)
Support Protocol 1: Glycerol/DMSO Shock of Mammalian Cells
9-7(1)
Alternate Protocol: High-Efficiency Transfection Using Calcium Phosphate-DNA Precipitate Formed in BES
9-8(1)
Transfection Using DEAE-Dextran
9-9(1)
Basic Protocol: General Procedure for DEAE-Dextran Transfection
9-9(1)
Alternate Protocol 1: Experiment: Transfection to Test Enzyme Structure/Activity Relationships
9-11(1)
Support Protocol: Charcoal Stripping of Fetal Bovine Serum
9-12(1)
Transfection by Electroporation
9-13(1)
Basic Protocol: Electroporation into Mammalian Cells
9-13(1)
Alternate Protocol: Electroporation into Plant Protoplasts
9-14(1)
Liposome-Mediated Transfection
9-15(1)
Basic Protocol: Transient Expression Using Liposomes
9-15(1)
Alternate Protocol: Stable Transformation Using Liposomes
9-16(1)
Selection of Transfected Mammalian Cells: Strategic Planning
9-16(1)
Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells
9-19(1)
Basic Protocol 2: Selectable Markers for Mammalian Cells
9-20(1)
Overview of Genetic Reporter Systems
9-24(1)
Design of Reporter Vectors
9-26(1)
In Vitro Reporter Assays
9-26(1)
In Vivo Reporter Assays
9-29(1)
Isotopic Assay for Reporter Gene Activity
9-33(1)
Basic Protocol 1: Chromatographic Assay for CAT Activity
9-33(1)
Alternate Protocol 1: In Situ Lysis of Cells for CAT Assay
9-35(1)
Alternate Protocol 2: Phase-Extraction Assay for CAT Activity
9-36(1)
Basic Protocol 2: Radioimmunoassay for Human Growth Hormone
9-37(1)
Nonisotopic Assays for Reporter Gene Activity
9-38(1)
Basic Protocol 1: Firefly Luciferase Reporter Gene Assay
9-38(1)
Alternate Protocol: Luciferase Assay in Freeze-Thaw-Lysed Cells
9-39(1)
Basic Protocol 2: Chemiluminescent βGalactosidase Reporter Gene Assay
9-40(1)
Overview of the Retrovirus Transduction System
9-41(1)
Retrovirus Life Cycle
9-41(1)
Replication-Incompetent Vectors
9-44(1)
Replication-Competent Vectors
9-46(1)
Packaging Lines and Virus Production
9-46(1)
Murine Retroviruses
9-50(1)
Avian Retroviruses
9-51(1)
Safety Issues
9-52(1)
Preparation of a Specific Retrovirus Producer Cell Line
9-52(1)
Basic Protocol 1: Introduction of a Retrovirus Vector into a Packaging Cell Line
9-52(1)
Basic Protocol 2: Determination of Viral Titer: Identification of Producer Clones Making High-Titer Virus
9-55(1)
Alternate Protocol: Rapid Evaluation of Producer Colonies
9-56(1)
Support Protocol: Xgal Staining of Cultured Cells
9-56(1)
Transient Transfection Methods for Preparation of High-Titer Retroviral Supernatants
9-57(1)
Basic Protocol 1: Transient Transfection of a Retrovirus Vector into 293 Cells
9-57(1)
Support Protocol: Growth and Storage of 293 Cells
9-58(1)
Basic Protocol 2: Infection of Adherent Cells with Retroviral Supernatant
9-59(1)
Alternate Protocol 1: Infection of Adherent Cells by Spin Infection
9-60(1)
Basic Protocol 3: Infection of Nonadherent Cells by Retroviral Supernatant
9-61(1)
Alternate Protocol 2: Infection of Nonadherent Cells by Cocultivation
9-62(1)
Alternate Protocol 3: Infection of Nonadherent Cells by Spin Infection
9-63(1)
Large-Scale Preparation and Concentration of Retrovirus Stocks
9-63(1)
Basic Protocol: Preparation of Virus Stock and Concentration by Centrifugation
9-64(1)
Alternate Protocol 1: Concentration by PEG Precipitation and Chromatography
9-65(1)
Alternate Protocol 2: Concentration Using Molecular-Weight- Cutoff Filters
9-65(1)
Detection of Helper Virus in Retrovirus Stocks
9-66(1)
Basic Protocol: Detection of Helper Virus Through Horizontal Spread of Drug Resistance
9-66(1)
Alternate Protocol 1: Proviral Rescue to Detect Replication-Competent Retrovirus
9-67(1)
Alternate Protocol 2: Reverse Transcriptase Assay to Detect Helper Virus
9-68(1)
Retrovirus Infection of Cells In Vitro and In Vivo
9-69(1)
Infection of Cells In Vitro
9-69(1)
Infection of Rodents In Vivo
9-70(1)
Analysis of Proteins
Overview of Protein Purification and Characterization
10-1(1)
Determination of Protein Concentration
10-8(1)
Basic Protocol 1: Using A280 to Determine Protein Concentration
10-8(1)
Basic Protocol 2: Using the Bradford Method to Determine Protein Concentration
10-9(1)
One-Dimensional SDS Gel Electrophoresis of Proteins
10-10(1)
Electricity and Electrophoresis
10-10(1)
Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method
10-11(1)
Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems
10-16(1)
Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers
10-18(1)
Alternate Protocol 3: Continuous SDS-PAGE
10-19(1)
Alternate Protocol 4: Casting and Running Ultrathin Gels
10-19(1)
Support Protocol 1: Casting Multiple Single-Concentration Gels
10-20(1)
Alternate Protocol 5: Separations of Proteins on Gradient Gels
10-22(1)
Support Protocol 2: Casting Multiple Gradient Gels
10-25(1)
Basic Protocol 2: Electrophoresis in Single-Concentration Minigels
10-27(1)
Support Protocol 3: Preparing Multiple Gradient Minigels
10-29(1)
Two-Dimensional Gel Electrophoresis Using the O'Farrell System
10-30(1)
Basic Protocol 1: First-Dimension (Isoelectric-Focusing) Gels
10-30(1)
Basic Protocol 2: Second-Dimension Gels
10-32(1)
Isoelectric Focusing of Very Basic and Very Acidic Proteins
10-34(1)
Alternate Protocol: Two-Dimensional Minigels
10-35(1)
Support Protocol: Solubilization and Preparation of Proteins in Tissue Samples
10-35(1)
Staining Proteins in Gels
10-36(1)
Basic Protocol 1: Coomassie Blue Staining
10-36(1)
Basic Protocol 2: Silver Staining
10-36(1)
Alternate Protocol: Nonammoniacal Silver Staining
10-37(1)
Basic Protocol 3: Rapid Silver Staining
10-38(1)
Support Protocol: Gel Photography
10-39(1)
Immunoblotting and Immunodetection
10-40(1)
Basic Protocol 1: Protein Blotting with Tank Transfer Systems
10-40(1)
Alternate Protocol 1: Protein Blotting with Semidry Systems
10-42(1)
Alternate Protocol 2: Blotting of Stained Gels
10-43(1)
Support Protocol 1: Reversible Staining of Transferred Proteins
10-44(1)
Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody
10-44(1)
Alternate Protocol 3: Immunoprobing with Avidin-Biotin Coupling to the Secondary Antibody
10-45(1)
Basic Protocol 3: Visualization with Chromogenic Substrates
10-46(1)
Alternate Protocol 4: Visualization with Luminescent Substrates
10-46(1)
Support Protocol 2: Stripping and Reusing Membranes
10-48(1)
Gel-Filtration Chromatography
10-48(1)
Basic Protocol 1: Desalting (Group Separation)
10-48(1)
Basic Protocol 2: Protein Fractionation
10-54(1)
Basic Protocol 3: Determination of Molecular Size
10-58(1)
Support Protocol: Column Calibration
10-60(1)
Ion-Exchange Chromatography
10-62(1)
Strategic Planning
10-62(1)
Basic Protocol 1: Batch Adsorption and Step-Gradient Elution with Increasing Salt Concentration
10-63(1)
Basic Protocol 2: Column Chromatography with Linear Gradient Elution
10-66(1)
Support Protocol 1: Test Tube Pilot Experiment to Determine Starting Conditions for Ion-Exchange Chromatography
10-68(1)
Immunoaffinity Chromatography
10-73(1)
Basic Protocol: Isolation of Soluble or Membrane-Bound Antigens
10-73(1)
Alternate Protocol 1: Batch Purification of Antigens
10-76(1)
Alternate Protocol 2: Low-pH Elution of Antigens
10-76(1)
Metal-Chelate Affinity Chromatography
10-76(1)
Basic Protocol: Native MCAC for Purification of Soluble Histidine-Tail Fusion Proteins
10-77(1)
Alternate Protocol 1: Denaturing MCAC for Purification of Insoluble Histidine-Tail Fusion Proteins
10-79(1)
Alternate Protocol 2: Solid-Phase Renaturation of MCAC-Purifie Proteins
10-81(1)
Support Protocol: NTA Resin Regeneration
10-81(1)
Reversed-Phase Isolation of Peptides
10-82(1)
Basic Protocol 1: Reversed-Phase Peptide Separation at the 5- to 500-pmol Level
10-82(1)
Basic Protocol 2: Reversed-Phase Peptide Separation at ≤5 pmol
10-83(1)
Support Protocol: Capillary HPLC System Assembly
10-84(1)
Purification of Recombinant Proteins by Epitope Tagging
10-86(1)
Basic Protocol 1: Immunoprecipitation of Epitope-Tagged Recombinant Proteins
10-86(1)
Immunoprecipitation
10-88(1)
Basic Protocol: Immunoprecipitation of Radiolabeled Antigen with Antibody-Sepharose
10-88(1)
Support Protocol: Preparation of Antibody-Sepharose
10-90(1)
Alternate Protocol 1: Immunoprecipitation of Radiolabeled Antigen with Anti-Ig Serum
10-91(1)
Alternate Protocol 2: Immunoprecipitation of Radiolabeled Antigen with Anti-Ig-Sepharose, Protein A- or G-Sepharose, or S. aureus Cells
10-92(1)
Alternate Protocol 3: Immunoprecipitation Using More Strongly Dissociating Lysis and Wash Buffers
10-93(1)
Alternate Protocol 4: Immunoprecipitation of Unlabeled Antigen with Antibody-Sepharose
10-93(1)
Synthesizing Proteins In Vitro by Transcription and Translation of Cloned Genes
10-94(1)
Metabolic Labeling with Amino Acids
10-96(1)
Safety Precautions for Working with 35S-Labeled Compounds
10-96(1)
Basic Protocol: Pulse-Labeling of Cells in Suspension with [35S]Methionine
10-96(1)
Alternate Protocol 1: Pulse-Labeling of Adherent Cells with [35S]Methionine
10-97(1)
Alternate Protocol 2: Pulse-Chase Labeling of Cells with [35S]Methionine
10-98(1)
Alternate Protocol 3: Long-Term Labeling of Cells with [35S]Methionine
10-99(1)
Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids
10-100(1)
Support Protocol: TCA Precipitation to Determine Label Incorporation
10-101(1)
Isolation of Proteins for Microsequence Analysis
10-102(1)
Basic Protocol 1: Determination of Amino Acid Sequence of Samples on PVDF Membranes
10-102(1)
Support Protocol: Preparation of Protein Samples for SDS-PAGE
10-104(1)
Basic Protocol 2: Determination of Internal Amino Acid Sequence from N-Terminally Blocked Proteins
10-104(1)
Immunology
Introduction
11-1(1)
Conjugation of Enzymes to Antibodies
11-3(1)
Basic Protocol: Conjugation of HRPO to Antibodies
11-3(1)
Alternate Protocol: Conjugation of Alkaline Phosphatase to Antibodies
11-4(1)
Enzyme-Linked Immunosorbent Assay (ELISA)
11-4(1)
Basic Protocol: Indirect ELISA to Detect Specific Antibodies
11-4(1)
Alternate Protocol 1: Direct Competitive ELISA to Detect Soluble Antigens
11-6(1)
Alternate Protocol 2: Antibody-Sandwich ELISA to Detect Soluble Antigens
11-8(1)
Alternate Protocol 3: Double Antibody-Sandwich ELISA to Detect Specific Antibodies
11-9(1)
Alternate Protocol 4: Direct Cellular ELISA to Detect Cell-Surface Antigens
11-11(1)
Alternate Protocol 5: Indirect Cellular ELISA to Detect Antibodies Specific for Surface Antigens
11-12(1)
Support Protocol 1: Criss-Cross Serial Dilution Analysis to Determine Optimal Reagent Concentrations
11-13(1)
Support Protocol 2: Preparation of Bacterial-Cell-Lysate Antigens
11-14(1)
Production of Monoclonal Antibody Supernatant and Ascites Fluids
11-15(1)
Basic Protocol 1: Production of a Monoclonal Antibody Supernatant
11-16(1)
Alternate Protocol 1: Large-Scale Production of Monoclonal Antibody Supernatant
11-16(1)
Alternate Protocol 2: Large-Scale Production of Hybridomas or Cell Lines
11-17(1)
Basic Protocol 2: Production of Ascites Fluid Containing Monoclonal Antibody
11-18(1)
Purification of Monoclonal Antibodies
11-20(1)
Basic Protocol: Purification Using Protein A-Sepharose
11-20(1)
Alternate Protocol 1: Alternative Buffer System for Protein A-Sepharose
11-20(1)
Alternate Protocol 2: Purification by Antigen-Sepharose and Anti-mouse-Ig-Sepharose
11-21(1)
Production of Polyclonal Antisera in Rabbits
11-22(1)
Basic Protocol: Intramuscular Immunization
11-22(1)
Alternate Protocol 1: Intradermal Immunization
11-24(1)
Alternate Protocol 2: Subcutaneous Immunization
11-24(1)
Alternate Protocol 3: Bleeding from the Ear Artery
11-24(1)
Purification of IgG Antibodies from Antiserum, Ascites Fluid, or Hybridoma Supernatant
11-25(1)
Basic Protocol: Precipitation of IgG with Ammonium Sulfate
11-25(1)
Alternate Protocol: Fractionation of IgG by Chromatography on DEAE-Affi-Gel Blue
11-26(1)
Selection of an Immunogenic Peptide
11-26(1)
Production of Antipeptide Antibodies
11-28(1)
Basic Protocol: Chemical Coupling of Synthetic Peptide to Carrier Protein Using MBS
11-29(1)
Alternate Protocol: Chemical Coupling of Synthetic Peptide to Carrier Protein Using Glutaraldehyde
11-29(1)
DNA-Protein Interactions
Introduction
12-1(1)
Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
12-3(1)
Basic Protocol: Preparation of Nuclear Extracts
12-3(1)
Support Protocol 1: Optimization of Nuclear Extraction
12-5(1)
Support Protocol 2: Preparation of the Cytoplasmic (S-100) Fraction
12-6(1)
Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
12-6(1)
Basic Protocol: Mobility Shift Assay
12-7(1)
Alternate Protocol 1: Competition Mobility Shift Assay
12-9(1)
Alternate Protocol 2: Antibody Supershift Assay
12-10(1)
Alternate Protocol 3: Multicomponent Mobility Shift Assays
12-10(1)
Methylation and Uracil Interference Assays for Analysis of Protein-DNA Interactions
12-11(1)
Basic Protocol 1: Methylation Interference Assay
12-11(1)
Basic Protocol 2: Uracil Interference Assay
12-13(1)
DNase I Footprint Analysis of Protein-DNA Binding
12-15(1)
Basic Protocol: DNase I Footprint Titration
12-15(1)
Support Protocol: Quantitation of Protein-Binding Equilibria by Densitometric and Numerical Analyses
12-18(1)
Alternate Protocol: DNase Footprinting in Crude Fractions
12-20(1)
UV Crosslinking of Proteins to Nucleic Acids
12-21(1)
Basic Protocol: UV Crosslinking Using a BrdU-Substituted Probe
12-21(1)
Alternate Protocol 1: UV Crosslinking Using a Non-BrdU-Substituted Probe
12-24(1)
Alternate Protocol 2: UV Crosslinking In Situ
12-24(1)
Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems
12-25(1)
Basic Protocol
12-25(1)
Alternate Protocol 1: Purification Using a Microcolumn
12-27(1)
Alternate Protocol 2: Purification Using Streptavidin-Agarose
12-28(1)
Detection, Purification, and Characterization of cDNA Clones Encoding DNA-Binding Proteins
12-28(1)
Basic Protocol: Screening aλgt11 Expression Library with Recognition-Site DNA
12-28(1)
Alternate Protocol: Denaturation/Renaturation Cycling of Dried Filters
12-30(1)
Support Protocol: Preparation of a Crude Extract from a Recombinant Lysogen to Characterize DNA-Binding Activity
12-31(1)
Analysis of DNA-Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes
12-32(1)
Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography
12-33(1)
Basic Protocol 1: Preparation of DNA Affinity Resin
12-33(1)
Alternate Protocol: Coupling the DNA to Commercially Available CNBr-Activated Sepharose
12-36(1)
Support Protocol 1: Purification of Oligonucleotides by Preparative Gel Electrophoresis
12-41(1)
Basic Protocol 2: DNA Affinity Chromatography
12-38(1)
Support Protocol 2: Selection and Preparation of Nonspecific Competitor DNA
12-40(1)
Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection
12-41(1)
Basic Protocol
12-41(1)
Support Protocol: Isolation and Analysis of Bound Oligonucleotides from Mobility Shift Gels
12-45(1)
Saccharomyces Cerevisiae
Introduction
13-1(1)
Preparation of Yeast Media
13-2(1)
Liquid Media
13-3(1)
Solid Media
13-5(1)
Strain Storage and Revival
13-7(1)
Basic Protocol: Preparation and Inoculation of Frozen Stocks
13-7(1)
Growth and Manipulation of Yeast
13-7(1)
Basic Protocol 1: Growth in Liquid Media
13-8(1)
Basic Protocol 2: Growth on Solid Media
13-8(1)
Basic Protocol 3: Determination of Cell Density
13-8(1)
Basic Protocol 4: Determination of Phenotype by Replica Plating
13-9(1)
Basic Protocol 5: Diploid Construction
13-9(1)
Basic Protocol 6: Sporulation of Diploid Cells
13-9(1)
Basic Protocol 7: Preparation and Dissection of Tetrads
13-10(1)
Support Protocol: Preparation of Dissecting Needles
13-12(1)
Alternate Protocol: Random Spore Analysis
13-13(1)
Mutagenesis of Yeast Cells
13-14(1)
Basic Protocol: Mutagenesis Using Ethyl Methanesulfonate
13-14(1)
Alternate Protocol: Mutagenesis Using UV Irradiation
13-16(1)
Yeast Cloning Vectors and Genes
13-16(1)
Plasmid Nomenclature
13-17(1)
Maps of Selected Plasmids and Genes
13-21(1)
Yeast Vectors for Expression of Cloned Genes
13-26(1)
Yeast Vectors and Expression Assays
13-28(1)
Basic Protocol 1: LacZ Fusion Vectors for Studying Gene Regulation
13-28(1)
Basic Protocol 2: Assay for β-galactosidase in Liquid Cultures
13-29(1)
Alternate Protocol: Screening for β-Galactosidase-Expressing Yeast Colonies Using a Filter Lift Assay
13-30(1)
Introduction of DNA into Yeast Cells
13-31(1)
Basic Protocol: Transformation Using Lithium Acetate
13-31(1)
Alternate Protocol 1: Spheroplast Transformation
13-32(1)
Alternate Protocol 2: Transformation by Electroporation
13-34(1)
Support Protocol: Preparation of Single-Stranded High-Molecular-Weight Carrier DNA
13-35(1)
Cloning Yeast Genes by Complementation
13-36(1)
Segregation of Plasmids from Yeast Cells
13-38(1)
Basic Protocol 1
13-38(1)
Basic Protocol 2: Plasmid Gap Repair
13-39(1)
Basic Protocol 3: Plasmid Shuffling
13-39(1)
Manipulation of Cloned Yeast DNA
13-41(1)
Basic Protocol 1: Integrative Transformation
13-41(1)
Gene Replacement Techniques
13-42(1)
Basic Protocol 2: Integrative Disruption
13-42(1)
Basic Protocol 3: One-Step Gene Disruption
13-42(1)
Alternate Protocol 1: PCR-Mediated One-Step Gene Disruption
13-43(1)
Basic Protocol 4: Transplacement
13-44(1)
Basic Protocol 5: Creating Modified Genes by One-Step Integrative Replacement
13-45(1)
Alternate Protocol 2: Creating Modified Genes by Transplacement
13-47(1)
Basic Protocol 6: Creation of Conditional Alleles by Copper-Inducible Double-Shutoff procedure
13-47(1)
Preparation of Yeast DNA
13-49(1)
Basic Protocol: Rapid Isolation of Plasmid DNA from Yeast
13-49(1)
Alternate Protocol: Rapid Isolation of Yeast Chromosomal DNA
13-50(1)
Preparation of Yeast RNA
13-51(1)
Basic Protocol: Preparation of Yeast RNA by Extraction with Hot Acidic Phenol
13-51(1)
Alternate Protocol 1: Preparation of RNA Using Glass Beads
13-52(1)
Alternate Protocol 2: Preparation of Poly(A)+RNA
13-53(1)
Preparation of Protein Extracts from Yeast
13-53(1)
Basic Protocol: Spheroplast Preparation and Lysis
13-53(1)
Support Protocol: Nuclei Preparation by Differential Centrifugation
13-55(1)
Alternate Protocol 1: Cell Disruption Using Glass Beads
13-55(1)
Alternate Protocol 2: Cell Disruption Using Liquid Nitrogen
13-56(1)
In Situ Hybridization and Immunohistochemistry
Introduction
14-1(1)
Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
14-2(1)
Basic Protocol: Paraformaldehyde Fixation and Paraffin Wax Embedding of Tissues and Embryos
14-2(1)
Alternate Protocol: PFA Fixation of Suspended and Cultured Cells
14-3(1)
Support Protocol 1: Perfusion of Adult Mice
14-4(1)
Support Protocol 2: Sectioning Samples in Wax Blocks
14-5(1)
Support Protocol 3: Preparation of Coated Slides
14-6(1)
Cryosectioning
14-7(1)
Basic Protocol: Specimen Preparation and Sectioning
14-7(1)
Support Protocol 1: Fixation of Cryosections for In Situ Hybridization
14-9(1)
Support Protocol 2: Tissue Fixation and Sucrose Infusion
14-11(1)
In Situ Hybridization to Cellular RNA
14-11(1)
Basic Protocol: Hybridization Using Paraffin Sections or Cells
14-11(1)
Alternate Protocol: Hybridization Using Cryosections
14-15(1)
Support Protocol 1: Synthesis of35S-Labeled Riboprobes
14-16(1)
Support Protocol 2: Synthesis of35S-Labeled Double-Stranded DNA Probes
14-17(1)
Detection of Hybridized Probe
14-17(1)
Basic Protocol 1: Film Autoradiography
14-18(1)
Basic Protocol 2: Emulsion Autoradiography
14-18(1)
Support Protocol: Preparation of Diluted Emulsion for Autoradiography
14-19(1)
Counterstaining and Mounting of Autoradiographed In Situ Hybridization Slides
14-19(1)
Basic Protocol: Giemsa Staining
14-19(1)
Alternate Protocol 1: Hematoxylin/Eosin Staining
14-21(1)
Alternate Protocol 2: Toluidine Blue Staining
14-21(1)
Alternate Protocol 3: Hoechst Staining
14-22(1)
Immunohistochemistry
14-22(1)
Basic Protocol 1: Immunofluorescent Labeling of Cells Grown as Monolayers
14-23(1)
Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells
14-23(1)
Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections
14-24(1)
Alternate Protocol 2: Immunofluorescent Labeling Using Streptavidin-Biotin Conjugates
14-25(1)
Alternate Protocol 3: Immunogold Labeling of Tissue Sections
14-25(1)
Alternate Protocol 4: Immunoperoxidase Labeling of Tissue Sections
14-26(1)
Alternate Protocol 5: Immunofluorescent Double-Labeling of Tissue Sections
14-28(1)
In Situ Hybridization and Detection Using Nonisotopic Probes
14-29(1)
Basic Protocol: Fluorescence In Situ Hybridization
14-29(1)
Amplification of Hybridization Signals
14-31(1)
Support Protocol 1: Amplification of Biotinylated Signals
14-31(1)
Support Protocol 2: Amplification of Signals from Digoxigenin-Labeled Probes
14-32(1)
Enzymatic Detection of Nonisotopically Labeled Probes
14-33(1)
Alternate Protocol 1: Enzymatic Detection Using Horseradish Peroxidase
14-33(1)
Alternate Protocol 2: Enzymatic Detection Using Alkaline Phosphatase
14-34(1)
In Situ Polymerase Chain Reaction and Hybridization to Detect Low-Abundance Nucleic Acids Targets
14-35(1)
Strategic Planning
14-35(1)
Basic Protocol 1: In Situ PCR (ISPCR) Amplification of DNA and RNA Targets with In Situ Reverse Transcription for RNA
14-38(1)
Alternate Protocol: One-Step Reverse Transcription and Amplification
14-42(1)
Basic Protocol 2: Hybridization and Detection of ISPCR-Amplified Target Material
14-43(1)
Support Protocol 1: Preparation of AES-Subbed Slides
14-45(1)
Support Protocol 2: Preparation of Specimens on Slides for ISPCR
14-45(1)
Support Protocol 3: Labeling Oligosaccharide Probes Using 33P
14-46(1)
The Polymerase Chain Reaction
Introduction
15-1(1)
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
15-3(1)
Direct DNA Sequencing of PCR Products
15-8(1)
Basic Protocol 1: Dideoxy Sequencing of Single-Stranded Products Generated by Asymmetric PCR
15-8(1)
Alternate Protocol 1: Generating Single-Stranded Template for Dideoxy Sequencing by Single-Primer Reamplification
15-9(1)
Alternate Protocol 2: Preparing Double-Stranded PCR Products for Dideoxy Sequencing
15-10(1)
Alternate Protocol 3: Dideoxy Sequencing of Single Strands Generated by λ Exonuclease Digestion of Double-Stranded PCR Products
15-10(1)
Basic Protocol 2: Labeling and Chemical Sequencing of PCR Products
15-11(1)
Alternate Protocol 4: Genomic Sequencing of PCR Products
15-12(1)
Quantitation of Rare DNA by PCR
15-13(1)
Enzymatic Amplification of RNA by PCR
15-16(1)
Basic Protocol: PCR Amplification of RNA Under Optimal Conditions
15-16(1)
Alternate Protocol 1: Avoiding Lengthy Coprecipitation and Annealing Steps
15-17(1)
Alternate Protocol 2: Introducing cDNA Directly into the Amplification Step
15-18(1)
Support Protocol: Rapid Precipitation of Crude RNA
15-18(1)
Ligation-Mediated PCR for Genomic Sequencing and Footprinting
15-19(1)
Basic Protocol: Ligation-Mediated Single-Sided PCR
15-19(1)
Support Protocol 1: Preparation of Genomic DNA from Monolayer Cells for DMS Footprinting
15-23(1)
Support Protocol 2: Preparation of Genomic DNA from Suspension Cells for DMS Footprinting
15-26(1)
Support Protocol 3: Preparation of Genomic DNA for Chemical Sequencing
15-27(1)
cDNA Amplification Using One-Sided (Anchored) PCR
15-29(1)
Basic Protocol 1: Amplification of Regions Downstream (3') of Known Sequence
15-29(1)
Basic Protocol 2: Amplification of Regions Upstream (5') of Known Sequence
15-32(1)
Molecular Cloning of PCR Products
15-34(1)
Basic Protocol: Generation of T-A Overhangs
15-34(1)
Alternate Protocol 1: Generation of Half-Sites
15-35(1)
Differential Display of mRNA by PCR
15-37(1)
Protein Expression
Introduction
16-1(1)
Overview of Protein Expression in E. coli
16-2(1)
General Strategy for Gene Expression in E. coli
16-3(1)
Specific Expression Scenarios
16-3(1)
Troubleshooting Gene Expression
16-4(1)
Expression Using the T7 RNA Polymerase/Promoter System
16-5(1)
Basic Protocol: Expression Using the Two-Plasmid System
16-5(1)
Alternate Protocol 1: Selective Labeling of Plasmid-Encoded Proteins
16-7(1)
Alternate Protocol 2: Expression by Infection with M13 Phage mGP1-2
16-8(1)
Introduction to Expression by Fusion Protein Vectors
16-9(1)
Solubility of the Expressed Protein
16-9(1)
Stability of the Expressed Protein
16-10(1)
Cleavage of Fusion Proteins to Remove the Carrier
16-10(1)
Enzymatic and Chemical Cleavage of Fusion Proteins
16-11(1)
Basic Protocol 1: Enzymatic Cleavage of Fusion Proteins with Factor Xa
16-11(1)
Support Protocol: Denaturing a Fusion Protein for Factor Xa Cleavage
16-12(1)
Alternate Protocol 1: Enzymatic Cleavage of Fusion Proteins with Thrombin
16-13(1)
Alternate Protocol 2: Enzymatic Cleavage of Matrix-Bound GST Fusion Proteins
16-13(1)
Alternate Protocol 3: Enzymatic Cleavage of Fusion Proteins with Enterokinase
16-14(1)
Basic Protocol 2: Chemical Cleavage of Fusion Proteins Using Cyanogen Bromide
16-15(1)
Alternate Protocol 4: Chemical Cleavage of Fusion Proteins Using Hydroxlamine
16-16(1)
Alternate Protocol 5: Chemical Cleavage of Fusion Proteins by Hydrolysis at Low pH
16-16(1)
Expression and Purification of Maltose-Binding Protein Fusions
16-17(1)
Basic Protocol: Construction, Expression, and Purification of MBP Fusion Proteins
16-18(1)
Support Protocol 1: Pilot Experiment to Characterize the Behavior of an MBP Fusion Protein
16-20(1)
Alternate Protocol: Purification of Fusion Proteins from the Periplasm
16-22(1)
Support Protocol 2: Purifying the Cleaved Protein by Ion Exchange Chromatography
16-22(1)
Support Protocol 3: Purifying the Cleaved Protein by Affinity Chromatography
16-23(1)
Expression and Purification of Glutathione-S-Transferase Fusion Proteins
16-24(1)
Expression and Purification of Thioredoxin Fusion Proteins
16-27(1)
Basic Protocol: Construction and Expression of a Thioredoxin Fusion Protein
16-27(1)
Support Protocol 1: E. coli Lysis Using a French Pressure Cell
16-29(1)
Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins
16-31(1)
Support Protocol 3: Purification of Thioredoxin Fusion Proteins by Heat Treatment
16-32(1)
Overview of the Baculovirus Expression System
16-33(1)
Baculovirus Expression System
16-33(1)
Post-Translational Modification of Proteins in Insect Cells
16-35(1)
Steps for Overproducing Proteins Using the Baculovirus Expression System
16-35(1)
Choosing a Baculovirus Transfer Vector: Choosing a Baculovirus DNA
16-35(1)
Choosing a Baculovirus DNA
16-38(1)
Reagents, Solutions, and Equipment for the Baculovirus Expression System
16-38(1)
Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses
16-39(1)
Basic Protocol 1: Maintenance and Culture of Insect Cells
16-40(1)
Basic Protocol 2: Cotransfection of Insect Cells Using Linearized Baculoviral DNA
16-41(1)
Alternate Protocol 1: Generation of Recombinant Baculovirus Using Wild-Type Baculoviral DNA
16-43(1)
Basic Protocol 3: Preparation of Baculovirus Stocks
16-45(1)
Basic Protocol 4: Titering Baculovirus Stocks Using Plaque Assay
16-47(1)
Expression and Purification of Recombinant Proteins Using the Baculovirus System
16-48(1)
Basic Protocol 1: Small-Scale Expression for Initial Analysis
16-49(1)
Support Protocol 1: Determining Time Course of Maximum Protein Production
16-50(1)
Support Protocol 2: Metabolic Labeling of Recombinant Proteins
16-51(1)
Basic Protocol 2: Large-Scale Production of Recombinant Proteins
16-52(1)
Basic Protocol 3: Purification of Recombinant Proteins Containing a Polyhistidine (6xHIS) Tag
16-53(1)
Alternate Protocol: Purification of Recombinant Proteins Containing a GST Tag
16-54(1)
Overview of Protein Expression in Mammalian Cells
16-56(1)
Viral-Mediated Gene Transfer
16-56(1)
Transient Expression
16-56(1)
Stable DNA Transfection
16-57(1)
Amplification of Transfected DNA
16-59(1)
Expression Vectors
16-59(1)
Choice of Expression System
16-60(1)
Troubleshooting
16-60(1)
Transient Expression of Proteins Using COS Cells
16-60(1)
Overview of the Vaccinia Virus Expression System
16-63(1)
Vaccinia Replication Cycle
16-63(1)
Effects of Vaccinia Infection
16-64(1)
Vaccinia Vector Expression System
16-65(1)
Steps For Expression of Genes Using Vaccinia Vectors
16-66(1)
Safety Precautions for Using Vaccinia
16-66(1)
Preparation of Cell Cultures and Vaccinia Virus Stocks
16-67(1)
Basic Protocol 1: Culture of Monolayer Cells
16-68(1)
Basic Protocol 2: Culture of Cells in Suspension
16-69(1)
Basic Protocol 3: Preparation of a Vaccinia Virus Stock
16-70(1)
Support Protocol 1: Titration of Vaccinia Virus Stocks by Plaque Assay
16-70(1)
Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts
16-71(1)
Basic Protocol 5: Preparation of an MVA Stock
16-72(1)
Support Protocol 2: Titration of MVA Stocks by Immunostaining
16-73(1)
Generation of Recombinant Vaccinia Viruses
16-75(1)
Basic Protocol 1: Transfection of Infected Cells with a Vaccinia Vector
16-75(1)
Support Protocol 1: Purification of Vaccinia Virus
16-79(1)
Support Protocol 2: Isolation of Vaccinia Virus DNA
16-81(1)
Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques
16-82(1)
Basic Protocol 3: Amplification of a Plaque
16-84(1)
Basic Protocol 4: Live Immunostaining of MVA Recombinants
16-85(1)
Support Protocol 3: Coating Plates with Concanavalin A
16-87(1)
Characterization of Recombinant Vaccinia Viruses and Their Products
16-87(1)
Basic Protocol 1: Detection of Vaccinia DNA Using PCR
16-88(1)
Basic Protocol 2: Detection of Vaccinia DNA Using Southern Blot Hybridization
16-90(1)
Basic Protocol 3: Detection of Vaccinia DNA Using Dot-Blot Hybridization
16-91(1)
Alternate Protocol: Detection of Expressed Protein by a dot-Blot Procedure
16-92(1)
Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting
16-93(1)
Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation
16-94(1)
Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase Hybrid System
16-95(1)
Basic Protocol 1: Liposome-Mediated Transfection Following Recombinant Vaccinia Virus (VTF7-3) Infection
16-95(1)
Basic Protocol 2: Coinfection with Two Recombinant Vaccinia Viruses
16-98(1)
Basic Protocol 3: Infection of OST7-1 cells with a Single Virus
16-100(1)
Basic Protocol 4: Gene Expression Using the Vote System
16-100(1)
Support Protocol: Detection of Expressed Protein Using Pulse Labeling
16-101(1)
Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
16-102(1)
Basic Protocol: Calcium Phosphate-Mediated Stable Transfection of NIH3T3 Cells with pTet-tTAk and Tetracyline-Regulated Target Plasmids
16-103(1)
Support Protocol: Analysis of Target Gene Protein Expression
16-107(1)
Analysis of Protein Phosphorylation
Introduction
17-1(1)
Overview of Protein Phosphorylation
17-1(1)
Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
17-3(1)
Basic Protocol: Labeling Cultured Cells with 32Pi and Lysis Using Mild Detergent
17-3(1)
Alternate Protocol: Lysis of Cells by Boiling in SDS
17-5(1)
Phosphoamino Acid Analysis
17-6(1)
Basic Protocol: Acid Hydrolysis and Two-Dimensional Electrophoretic Analysis of Phosphoamino Acids
17-6(1)
Alternate Protocol: Alkali Treatment to Enhance Detection of Tyr- and Thr-Phosphorylated Proteins Blotted onto Filters
17-9(1)
Analysis of Phosphorylation of Unlabeled Proteins
17-10(1)
Basic Protocol 1: Immunoblotting with Anti-Phosphotyrosine Antibodies and Detection Using [125I]Protein A
17-10(1)
Alternate Protocol: Detection of Bound Antibodies by Enhanced Chemiluminescence (ECL)
17-11(1)
Basic Protocol 2: Identification of Phosphorylated Proteins by Phosphatase Digestion
17-12(1)
Detection of Phosphorylation by Enzymatic Techniques
17-13(1)
Basic Protocol 1: Digestion of Phosphoproteins with Nonspecific Acid Phosphatases
17-14(1)
Alternate Protocol 1: Digestion of Phosphoproteins with Nonspecific Alkaline Phosphatase
17-15(1)
Basic Protocol 2: Digestion of Phosphoproteins with Protein Serine/Threonine Phosphatases
17-15(1)
Alternate Protocol 2: Digestion of Phosphoproteins with Protein Tyrosine Phosphatases
17-16(1)
Support Protocol: Measurement and Identification of Released 32P
17-16(1)
Assays of Protein Kinases Using Exogenous Substrates
17-17(1)
Strategic Planning
17-17(1)
Basic Protocol 1: Assay for Cyclic Nucleotide-Dependent Protein Kinases
17-19(1)
Basic Protocol 2: Assay for Protein Kinase C Isoforms
17-20(1)
Basic Protocol 3: Assay for Casein Kinases Using β-Casein
17-20(1)
Alternate Protocol: Assay for Casein Kinases Using a Peptide Substrate
17-21(1)
Basic Protocol 4: Assay for Ca2+/Calmodulin-Dependent Kinases
17-22(1)
Basic Protocol 5: Assay for Tyrosine Kinases
17-23(1)
Basic Protocol 6: In-Gel Protein Kinase Assays
17-24(1)
Support Protocol 1: Preparing a Cell Lysate for Kinase Assays
17-25(1)
Support Protocol 2: TCA Precipitation to Determine Incorporation of Radioactivity
17-25(1)
Support Protocol 3: Adsorption onto p81 Phosphocellulose Paper
17-26(1)
Permeabilization Strategies to Study Protein Phosphorylation
17-28(1)
Basic Protocol 1: Analysis of Protein Phosphorylation in Permeabilized Cells
17-28(1)
Intact Cell Sample Preparation for Electrophoretic Analysis of Protein Phosphorylation
17-31(1)
Alternate Protocol 1: Intact Cell Sample Preparation for SDS-PAGE
17-31(1)
Alternate Protocol 2: Intact Cell Sample Preparation for Isoelectric Focusing
17-32(1)
Sequence Similarity Searching Using the Blast Family of Programs
Sequence Similarity Searching Using the Blast Family of Programs
18-1(1)
Accessing Blast Programs and Documentation
18-1(1)
Introduction to Blast
18-2(1)
Examples of Blast Searches
18-6(1)
Searching Strategies
18-19(1)
Appendix A: Blast Parameters
18-22(1)
Appendix B: Sequence Identifier Syntax
18-23(1)
Analysis of Protein Interactions
Introduction
19-1(1)
Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
19-5(1)
Basic Protocol 1: Characterizing a Bait Protein
19-5(1)
Basic Protocol 2: Performing an Interactor Hunt
19-13(1)
Alternate Protocol 1: Rapid Screen for Interaction Trap Positives
19-20(1)
Alternate Protocol 2: Performing a Hunt by Interaction Mating
19-22(1)
Support Protocol 1: Preparation of Protein Extracts for Immunoblot Analysis
19-24(1)
Support Protocol 2: Preparation of Sheared Salmon Sperm Carrier DNA
19-25(1)
Affinity Purification of Proteins Binding to GST Fusion Proteins
19-26(1)
Basic Protocol: GST Fusion Protein-Affinity Purification
19-26(1)
Support Protocol: Preparation of E. coli Extract
19-28(1)
Phage-Based Expression Cloning to Identify Interacting Proteins
19-29
Strategic Planning
19-29
Basic Protocol: Interaction cloning
19-31
Appendices
A1 Reagents and Solutions A1-1
A2 Standard Measurements and Data A2-1
Useful Measurements and Data A2-1
Characteristics of Nucleic Acids A2-6
Radioactivity A2-12
Centrifuges and Rotors A2-15
A3 Commonly Used Techniques in Biochemistry and Molecular Biology A3-1
3A Autoradiography A3-1
3B Silanizing Glassware A3-3
3C Dialysis and Ultrafiltration A3-4
3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy A3-10
3E Introduction of Restriction Enzyme Recognition Sequences by Silent Mutation A3-12
3F Techniques for Mammalian Cell Tissue Culture A3-14
3G Safe Use of Radioisotopes A3-22
3H Statistics for the Molecular Biologist: Group Comparisons A3-33
A4 Selected Suppliers of Reagents and Equipment A4-1
A5 References A5-1
Index

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Reviews for Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology (9780471329381)