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

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

9780471329381

047132938X

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