Cloning, Gene Expression and Protein Purification

Introductory Unit Introductory Lecture - Introduction to the Biochemical Laboratory Theory: Course Description Theory: "Central Dogma of Molecular Biology" Theory: Laboratory Safety Theory: The Scientific Method: Surviving Recipe Mentality Theory: Proactive Troubleshooting Theory: Introduction to the Biotechnology Laboratory Theory: Error Analysis and Assay Sensitivity Theory: Treatment of Analytical Data Theory: Concentration and Temperature Effects on pKa Introductory Lab 1 - Basic Biochemical Techniques I: Pipet Calibration and Solution Preparation Process: Pipets Introductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration Determinations Process: AMP and Tryptophan Absorbance Spectra; Sample Calculations Theory: Absorption Data for the Nucleoside Monophosphates Process: Absorption Spectra Data for the Aromatic Amino Acids at pH 6; UV Absorption Characteristics of the Aromatic Amino Acids. Selected Extinction Coefficients Process: BCA Assay Sample Data Innov.: Measurement of Protein in 20 Seconds Part I - Nucleic Acids & Cloning Unit 1 Lecture 1 - DNA Isolation Theory: Subcloning Procedure Innov.: The pET Bacterial Plasmid System (Novagen) Lab 1.1 - Media Preparation; Bacterial Growths; Plasmid Minipreps; HindIII Digestion of DNA, Commercial Bacteriophage h DNA BstEII Digest Size Standards Process: pUR278 and p2D Restriction Maps Process: Cloning the myo-3 Gene from C. elegans and Construction of an Expression Vector Process: C. elegans myo-3 Gene in pUR288 Vend. Lit.: Restriction Enzymes HindIII and BstEII; h DNA Digests Process: Phage h BstEII Digest Lab 1.2 - Agarose Gel Electrophoresis Exercises: Restriction Mapping Unit 2 Lecuture 2 - Construction of Recombinant Plasmids Innov.: Protecting and Manipulating Large DNA Substrates Innov.: Yeast of Burden--Yoking the YAC Lab 2.1 - Extraction and Cleanup of DNA Bands Cut from Agrose Gels, Quantitation of Yields and Ligation of myo-3 HindIII DNA Insert Fragment into Linearized B-gal Plasmid DNA Vend. Lit.: Gibco BRL (TM) T4 DNA Ligase Vend. Lit.: DNA Purification Kit (NaI/Glass Bead Method) Alt. App.: The Use of B-Agarase to Recover DNA from Gel Slices Alt. App.: GELase TM Unit 3 Lecture 3 - The Polymerase Chain Reaction Innov.: Polymerase Chain Reaction Used for Antigen Detection; Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA Conjugates Lab 3.1 - Polymerase Chain Reaction Test for myo-3 Gene Insert Orientation Unit 4 Lecture 4 - Transcription of Genomic DNA & Analysis of the Resulting mRNAs Alt. App.: Isolation of Total RNA from E. coli Cells Alt. App.: Promega TM PolyATractTM System 1000 Alt. App.: Electrophoresis and Northern Blotting of RNA Unit 5 Lecture 5 - Transformation and Gene Expression Innov.: How Cells Respond to Stress Lab 5.1 - Preparation of Fresh Transformation - Competent Cells Alt. App.: Ultracomp TM Transformation Kit Lab 5.2 - Colony Immunoblotting to Screen for Transformants Alt. App.: The QIAexpressionist, QIAGENTM Unit 6 Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and Probing Innov.: Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity than 32P-Based Hybridization Lab 6.1 - Labeling of DNA and Probe Construction from Cloned C. elegans myo-3 Gene; Quantitation of DNA Concentration Vend. Lit.: Digoxigenin Labeling of DNA: Genius TM Nucleic Acid Labeling System Lab 6.2 - Isolation of C. elegans Genomic DNA, Quantitation of DNA Concentration, and Digestion to Extract the myo-3 Gene Lab 6.3 - Southern Blotting Part 2 - Protein Purification Unit 7 Lecture 7 - Protein Purification Theory: Preparation and Handling of Biological Macromolecules for Crystallization Theory: Solution STructure of Biomacromolecules in Ionic Solutions Theory: Solubility as a Function of Protein Structure and Solvent Components Theory: Dominant Forces in Protein Folding Alt. App.: Hydrophobic Interaction Chromatography Alt. App.: Centriprep Microconcentrators for Small Volume Concentrations; Centricon-3 and 100 Lab 7.1 - The Protein Purifier: A Learning Aid from Pharmacia Lab 7.2 - Induction and Purification of B-Galactosidase Fusion Protein from Bacteria Lab 7.3 - Gel Filtration of Molecular Weight Standards and Protein Fractionation Process: Gel Filtration and Chromatography Vend. Lit.: Sephadex and Sephacryl Vend. Lit.: Sigma TM Gel Filtration Molecular Weight Markers Lab 7.4 - Mciroplate B-Galactosidase Assay to Determine Fractions Containing Fusion Protein; MW Determination Process: Time Course Assay of B-Galactosidase Vend. Lit.: B-Galactosidase Substrates Innov.: Luminescent Reporter Gene Assays for Luciferase and B-Galactosidase Using a Liquid Scintillation Counter Lab 7.5 - Ion Exchange Column Chromatography Process: Ion Exchange Chromatography Theory: The Isoelectric Point: Protein Charge Neutrality at a Particular pH Alt. App.: Ion-Pair Chromatography Alt. App.: HPLC: Ion Exchange and Reverse Phase Methods; Literature Sources Lab 7.6 - Affinity Chromatography and Microplate B-Galactosidase Assays to Determine Fractions Containing Fusion Protein Process: Affinity Chromatography Process: Affinity Chromatography: One Step Purification of Hybrid Proteins Carrying Fused B-Galactosidase Activity Lab 7.7 - BCA Protein Concentration Assays and B-Galactosidase Assays to Construct an Enzyme Purification Table Unit 8 Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size Determination Process: Discontinuous Gel Electrophoresis & Protein Size Determination Lab 8.1 - Discontinuous SDS Gel Electrophoresis Unit 9 Lecture 9 - Immunochemical Techniques Innov.: Immunochemical Techniques Innov.: The Enzyme Linked Immunosorbent Assay (ELISA) Innov.: How the Immune System Learns About Self Innov.: Making Monoclonal Antibodies That Won't Fight Back Lab 9.1 - Western Blotting Process: Immunoblotting Process: Western Blots Using Stained Protein Gels Unit 10 Lecture 10 - Combinatorial Biochemical Technology Innov.: Examples of Combinatorial Techniques Innov.: Making Antibody Fragments Using Phage Display Libraries Innov.: Building a Better Enzyme Innov.: The ImmunoZAP Cloning and Expression System Appendices Part 1 Terms List Part 2 Terms List Laboratory Reagents Abbreviations List Copyright Acknowledgements Suggested Schedule Supplies Required Index (Abbreviations: Innov., Innovation/Insight Theory, Theory/Principles Process, Process Rationale Vend. Lit., Vendor Literature Alt. App., Alternative Approach