 |
Molecular biology techniques : a classroom laboratory manual / by Susan Carson, Heather Miller, D. Scott Witherow. — 3rd ed. — Oxford ;Waltham, MA : Academic, 2012. – (58.21/R649/3rd ed.) |
Contents
CONTENTS
Preface
About the Authors
Acknowledgements
Note to Instructors
Instrumentation
Nomenclature
Introduction
Conceptual Outline for Experiments
Experimental Procedures
Laboratory Safety
General Operating Procedures
Part 1 manipulation of DNA
Reference
Lab Session 1 Getting Oriented: Practicing with Micropipettes
Station Checklist
Micropipetting
Micropipetting Self-Test
Laboratory Exercise: BSA Serial Dilutions and Nitrocellulose Spot Test
Preparing BSA Dilutions
Performing a Nitrocellulose Spot Test
Discussion Questions
Lab Session 2 Purification and Digestion of Plasmid (Vector) DNA
Introduction to Plasmid Purification
Alkaline Lysis
Silica Adsorption
DNA Quantification
Introduction to Expression Vectors
Principles of Gene Expression
Expression Vectors
Orientation and Reading Frame
Orientation
Reading Frame
Laboratory Exercises
Alkaline Lysis and Silica Adsorption Protocol
DNA Quantification
Restriction Digestion of Expression Vector DNA pET-41 a， a GST Fusion Protein Vector
Discussion Questions
Reference
Lab Session 3 PCR Amplification of egfp and Completion of Vector Preparation
Introduction
What is the Polymerase Chain Reaction (PCR)?
Why Clone by PCR?
TA Cloning
PCR Cloning by Incorporation of Restriction Sites
Cloning Synthetic Genes
Laboratory Exercises
PCR Amplification of egfp from the pEGFP-N1 Plasmid
PCR Protocol
Clean-up of Digested pET-41 a Vector
Agarose Gel Electrophoresis
Discussion Questions
References
Lab Session 4 Preparation of Insert DNA (egfp) PCR Product
Check PCR Reactions on an Agarose Gel
Spin Column Cleanup of PCR Product
Quantification of egfp PCR Product
Restriction Digestion of egfp PCR Product
Removing Enzymes and Cleaning Digested DNA Using a Spin Column
Discussion Questions
Lab Session 5 DNA Ligation and Transformation of Escherichia coli
Introduction
Ligation
Transformation
Laboratory Exercises
Ligations and Ligation Controls
Divalent Cation-Mediated Transformation
Electrophoresis of Ligation Reactions
Discussion Questions
Reference
Lab Session 6 Colony Hybridization
Lab Session 6A Interim Laboratory Session
Introduction
Laboratory Exercises
Counting Transformants and Replica Plating
Replica Plating
Lab Session 6B Colony Hybridization: Monoclonal Antibody Probe
Introduction
Laboratory Exercises
Colony Hybridization with an α-EGFP Monoclonal Antibody Probe: Part 1
Lab Session 6 Discussion Questions
Lab Session 7 Characterization of Recombinant Clones: Part 1
Lab Session 7A Completion of Colony Hybridization with a Monoclonal Antibody Probe
Introduction
Laboratory Exercise
Colony Hybridization with an α-EGFP Monoclonal Antibody Probe: Part 2
Lab Session 7B PCR Screening
Introduction
Laboratory Exercise
Polymerase Chain Reaction Screen for Recombinant Clones
Lab Session 7C Prepare Fresh Replica Plate
Lab Session 7 Discussion Questions
Lab Session 8 Characterization of Recombinant Clones: Part 2
Lab Session 8A Interim Laboratory Session
Laboratory Exercise
Inoculate Cultures for Minipreps
Lab Session 8B Analysis of PCR Screen Results
Introduction
Laboratory Exercise
Gel Electrophoresis and Analysis of PCR Samples from Last Week
Lab Session 8C Isolation of Miniprep DNA from Potential Transformants
Introduction
Laboratory Exercise
Isolation of Miniprep DNA from Potential Transformants
Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge
Lab Session 8D Visualization of Green Fluorescent Protein: Part 1
Introduction
Laboratory Exercise
Green Fluorescence Assay and Preparation of a Fresh Master Plate
Lab Session 8 Discussion Questions
Lab Session 9 Characterization of Recombinant Clones: Part 3
Lab Session 9A Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants)
Introduction
Laboratory Exercise
Restriction Enzyme Analysis of Miniprep DNA
Lab Session 9B Visualization of Green Fluorescent Protein: Part 2
Introduction
Laboratory Exercise
Visualization of Clones Expressing the Enhanced Green Fluorescent Protein on IPTG Plates
Lab Session 9C Computational Analysis of DNA Sequence from a Positive Clone: Part 1
Introduction
Laboratory Exercise
References
Lab Session 9 Discussion Questions
Lab Session 10 Computational Analysis of DNA Sequence from a Positive Clone: Part 2
Introduction
Laboratory Exercise
Discussion Questions
Reference
PART 3 - Expression， Detection and Purification of Recombinant Proteins from Bacteria
Lab Session 11 Expression of Fusion Protein from Positive Clones， SDS-PAGE and Western Blot: Part 1
Lab Session 11A Interim Laboratory Session
Laboratory Exercise
Inoculate Cultures for SDS-PAGE
Lab Session 11B Expression of Fusion Protein from Positive Clones， SDS-PAGE and Western Blot
Introduction
Laboratory Exercise
SDS-PAGE and Western Blot: Part 1
Reference
Lab Session 11 Discussion Questions
Lab Session 12 Expression of Fusion Protein from Positive Clones， SDS-PAGE and Western Blot: Part 2
Introduction
Laboratory Exercises
SDS-PAGE and Western Blot: Part 2
Replica Plate Positive Clone
Discussion Questions
Lab Session 13 Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column
Lab Session 13A Interim Laboratory Session
Laboratory Exercise
Inoculate Cultures for Protein Purification
Lab Session 13B Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column
Introduction
Laboratory Exercises
Growing Bacterial Suspension Cultures for Fusion Protein Purification
Harvesting IPTG-Induced Cultures
Breaking Open Bacterial Cells
Removing Insoluble Debris from the Crude Homogenate
Purifying Protein by Affinity Chromatography
Lab Session 13 Discussion Questions
Lab Session 14 Analysis of Purification Fractions
Lab Session 14A Analysis of Purification Fractions
Introduction
Laboratory Exercises
SDS-PAGE of Purified Fusion Protein
Fluorescence Analysis of Affinity Purification
Lab Session 14B Replica Plate Positive Clone
Lab Session 14 Discussion Questions
PART 4 Analysis of mRNA levels
Challenges of Working with RNA
References
Lab Session 15 Total RNA Purification
Lab Session 15A Interim Laboratory Session
Laboratory Exercise
Inoculate Cultures for RNA Purification
Lab Session 15B Total RNA Purification
Introduction
Laboratory Exercises
Purification of Total RNA
DNase Digestion
Quantification of RNA
References
Lab Session 15 Discussion Questions
Lab Session 16 Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 1
Introduction
Reverse Transcription
Quantitative PCR
Laboratory Exercises
Reverse Transcription
Quantitative PCR (qPCR)
Discussion Questions
Reference
Lab Session 17 Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 2
Introduction
Laboratory Exercise
Relative Quantification of gst::egfp Levels
References
Discussion Questions
Lab Session 18 Analysis ofgst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1
Introduction
Laboratory Exercises
Reverse Transcription (RT)
Semi-Quantitative PCR
Discussion Questions
Lab Session 19 Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 2
Introduction
Laboratory Exercises
Agarose Gel Electrophoresis
Quantification
Discussion Questions
Reference
Appendix 1: Equipment
Appendix 2: Prep List
Appendix 3: Preparation of Competent E. coli Cells
Appendix 4: Pre-Lab Questions
Index
