Note: Front Cover -- Molecular Biology Techniques: A Classroom Laboratory Manual -- Copyright Page -- Contents -- Preface -- About the Authors -- Acknowledgements -- Note to Instructors -- Instrumentation -- Nomenclature -- Introduction -- Conceptual Outline for Experiments -- Experimental Procedures -- Laboratory Safety -- General Operating Procedures -- 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-41a, 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-41a 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 -- 2 - Screening Transformants -- 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 -- 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 -- 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 of gst::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 -- Shared Equipment -- Appendix 2: Prep List -- Notes to Prep Staff -- Plasmids and E. coli Host Strains -- Antibiotics -- Aliquots -- Bacterial Waste -- Autoclaving -- General Lab Preparation -- Students -- Supplies and Reagents -- Supplies and Reagents for General Use -- Recipes for General Use -- LAB SESSION 1 -- BSA Serial Dilutions and Nitrocellulose Spot Test -- Recipes -- LAB SESSION 2 -- Purification and Digestion of Plasmid (Vector) DNA -- LAB SESSION 3 -- PCR Amplification of egfp from pEGFP-N1 -- Clean-up and Visualization of Digested pET-41a Vector -- Recipes -- LAB SESSION 4 -- Preparation of Insert DNA (egfp) PCR Product -- LAB SESSION 5 -- DNA Ligation and Transformation of Escherichia coli -- LAB SESSION 6 -- Interim Lab -- LAB SESSION 6 -- Colony Hybridization: Monoclonal Antibody Probe -- Recipes -- LAB SESSION 7 -- Characterization of Recombinant Clones -- Recipes -- Chloronaphthol Stock Solution -- Peroxide Stain -- LAB SESSION 8 -- Interim Lab -- LAB SESSION 8 -- Characterization of Recombinant Clones: Part 2 -- LAB SESSION 9 -- Characterization of Recombinant Clones: Part 3 -- LAB SESSION 10 -- Computational Analysis of DNA Sequence from a Positive Clone: Part 2 -- LAB SESSION 11 -- Interim Lab -- LAB SESSION 11 -- Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1 -- Recipes -- LAB SESSION 12. , Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2.