Foreword
Acknowledgments
Note to Instructors
INTRODUCTION Conceptual Outline for Experiments
I. PROCEDURES
II. LABORATORY SAFETY
III. MICROPIPETTES
PART I Manipulation of DNA
LAB SESSION 1 Culturing Escherichia coli for Plasmid Isolation
I. INTRODUCTION
II. ALPHA COMPLEMENTATION
III. LABORATORY EXERCISES
A. Preparation of Solid Media
B. Making Stab Cultures
C. Streaking out Plasmid Clones on LB/Carb Plates
D. Growth Curve of Escherichia coli
E. Growing Bacterial Suspension Cultures for Plasmid Preparations
REFERENCES
SOLUTIONS
LAB SESSION 2 Growth Curve of Escherichia coli
I. INTRODUCTION
II. PLASMID COPY CONTROL
III. LABORATORY EXERCISES
A. Examination of Clones
B. Escherichia coli Growth Curve
C. Preparation of Solutions for Large-Scale Plasmid DNA Preps
D. Inoculation of Cultures for Large-Scale Plasmid DNA Preps
SOLUTIONS
LAB SESSION 3A Large-Scale Preparation of Plasmid DNA: Alkaline Lysis
I. INTRODUCTION
II. LABORATORY EXERCISES
Alkaline Lysis of Large-Scale Cultures
REFERENCES
LAB SESSION 3B Purification of Plasmid DNA
Using Columns
LABORATORY EXERCISES
REFERENCES
SOLUTIONS
LAB SESSION 3C Purification of Plasmid DNA Using Cesium Gradients
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Beckman TLV-100 Table Top Ultracentrifuge with a Vertical Rotor
B. XL-90 Centrifuge
REFERENCES
SOLUTIONS
LAB SESSION 3D Precipitation of Plasmid DNA Using Polyethylene Glycol
I. INTRODUCTION
II. LABORATORY EXERCISES
REFERENCES
SOLUTIONS
LAB SESSION 4 Preparation of Vector DNA
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Ethidium Bromide Spot Test
B. Preparation of HindIII-Cut, Phosphatased Vector
C. Gel Electrophoresis
D. Electrophoresis of pUR288
E. Shrimp Alkaline Phosphatase Treatment of pUR288
F. Overnight Cultures for Transformation-Competent Cells
SOLUTIONS
APPENDIX
LAB SESSION 5 Transformation of Escherichia coli
I. INTRODUCTION
II. LIGATION
III. LABORATORY EXERCISES
A. Preparation of Frozen, Transformation-Competent Cells
B. Test Ligations of pUR288
C. Electrophoresis of Ligation Products
D. Transformation
REFERENCES
SOLUTIONS
LAB SESSION 6 Preparation and Ligation of Insert DNA
I. INTRODUCTION
II. LIGATIONS
III. LABORATORY EXERCISES
A. Analysis of Vector Preparation
B. Isolation of myo-3 DNA from Agarose
C. Ligations
REFERENCES
SOLUTIONS
LAB SESSION 7A Transformation of Escherichia coli with Ligated DNA; Labeling a DNA Probe
I. INTRODUCTION
II. ELECTROPORATION
III. LABORATORY EXERCISES
A. Divalent Cation-Mediated Transformation
B. Electroporation
C. Electrophoresis of Ligation Reactions
D. Random Primed DNA Labeling with Digoxigenin-11-dUTP
REFERENCES
MATERIALS AND SOLUTIONS
LAB SESSION 7B Labeling DNA with Radioactive Nucleotides
I. INTRODUCTION
II. SAFETY PRECAUTIONS FOR WORKING WITH RADIOACTIVITY
III. LABORATORY EXERCISES
A. Random Primer Labeling of DNA Using [(32)P]dCTP
B. Using Spin Columns
MATERIALS AND SOLUTIONS
LAB SESSION 8 DNA Probe Quantitation and Replica Plating
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Estimating the Yield of Digoxigenin-Labeled Probe
B. Making Replica Plates
MATERIALS AND SOLUTIONS
LAB SESSION 9 Colony Hybridization I: Working with a DNA Probe
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Colony Hybridizations: Transfer of Colonies to Nylon Membranes
B. Prehybridization
C. Hybridization with the Digoxigenin-11-dUTP-Labeled Probe
D. Prehybridization for [(32)P]dCTP-Labeled DNA Probe
E. DNA/DNA Hybridization: Adding [(32)P]dCTP-Labeled DNA Probe
REFERENCES
SOLUTIONS
APPENDIX
LAB SESSION 10 Restriction Map of Plasmid p2D; Colony Hybridization
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Restriction Enzyme Reactions
B. Colony Hybridization with a DNA Probe: Washing Filters Probed with
Digoxigenin-11-dUTP-Labeled myo-3 Fragment
C. Colony Hybridization with a DNA Probe: Washing Filters Probed with
[(32)P]dCTP-Labeled myo-3 Fragment
D. Gel Electrophoresis of Restriction Digests
REFERENCES
MATERIALS AND SOLUTIONS
LAB SESSION 11 Screening Colonies with Monoclonal Antibodies
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Colony Hybridizations: Monoclonal Antibody Probe (Day 1)
B. Set-up for Alkaline Lysis Minipreps
REFERENCES
SOLUTIONS
LAB SESSION 12 Isolation and
Characterization of Miniprep DNA from Potential Transformants
I. INTRODUCTION
II. POLYMERASE CHAIN REACTION
III. LABORATORY EXERCISES
A. Colony Hybridizations: Monoclonal Antibody Probe (Day 2)
B. Completion of Alkaline Lysis Minipreps
C. Restriction Enzyme Analysis of Miniprep DNA
D. Polymerase Chain Reaction Screen for Recombinant Clones
REFERENCES
SOLUTIONS
APPENDIX Mapping Exercise: Analysis of Miniprep DNA Cut with Restriction Enzyme EcoRV
LAB SESSION 13 Titering Bacteriophage Lambda
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Titering Bacteriophage Lambda
B. Analysis of Polymerase Chain Reactions
C. Overnight Cultures for Electrophoresis of Proteins
REFERENCES
SOLUTIONS
PART II Expression, Detection, and Purification of Recombinant Proteins from
Bacteria
LAB SESSION 14 Expression of Fusion Protein in Bacterial Minipreps and Preparation of
Sodium Dodecyl Sulfate-Polyacrylamide Gels
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Growing Bacterial Suspension Cultures for Gel and Immunoblot Analysis
B. Pouring Stacking Gels for SDS-Polyacrylamide Slab Gels
C. Denaturing Protiens with Sodium Dodecyl Sulfate
D. Preparing Molecular Weight Standards
E. Loading Samples on the Gel
F. Electrophoresis
G. Stopping Electrophoresis
H. Staining the Gel
I. Drying the Gel
J. Inoculating Cultures for Large-Scale Purification
K. Calculations
SOLUTIONS
APPENDIX Pouring SDS-Polyacrylamide Gels
LAB SESSION 15 Extraction of Recombinant
Protein from Escherichia coli
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Growing Bacterial Suspension Cultures for Fusion Protein Purification
B. Harvesting IPTG-Induced Expression Vector Cultures
C. Breaking Open Bacterial Cells
D. Removing Insoluble Debris from the Crude Homogenate
E. Precipitating Protein with Ammonium Sulfate
F. Purifying Protein by Affinity
Chromatography
REFERENCES
SOLUTIONS
Introduction to Lab Session 16: Quantitation of the Yield and Purity of Recombinant
Beta-Galactosidase Fusion Proteins
LAB SESSION 16A Protein Determinations
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Preparing Standard BSA Dilutions
B. Performing a Nitrocellulose Spot Test
C. Preparing BSA Standard Curve in Preparation for Bradford Assays
D. Assaying the Unknown: Subjecting Protein Samples to the Bradford Assay
E. Calculating Protein Concentrations
REFERENCES
SOLUTIONS
APPENDIX Sample Calculations of Protein
Concentrations
LAB SESSION 16B Beta-Galactosidase Assays
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Performing a Practice Beta-Galactosidase Assay
B. Performing the Beta-Galactosidase Assays with Fusion Proteins
C. Calculations of Specific Activity
D. Presenting Data and Calculations
REFERENCES
SOLUTIONS
APPENDIX Performing Protein and Enzyme Assays with an ELISA Plate Reader
LAB SESSION 17 Immunological Analysis of the Purified Fusion Proteins
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Performing SDS-PAGE in Preparation for Immunoblotting
B. Electrophoretic Transfer of Proteins to Nitrocellulose
C. Staining the Blot with Protein Dye, Amido Black
REFERENCES
SOLUTIONS
LAB SESSION 18 Immunoblot Development
I. INTRODUCTION
II. LABORATORY EXERCISES
A. Incubating the Blot with Primary Antibody (Monoclonal Antibody 5-6)
B. Incubation with Goat Anti-Mouse Peroxidase
C. Chemiluminescent Peroxidase Reaction
III. QUESTIONS
MATERIALS AND SOLUTIONS
APPENDIX Colorimetric