Cloning, Gene Expression and Protein Purification Experimental Procedures and Process Rationale

The central purpose of this combination lecture/laboratory manual is to present detailed protocols for the multi-step process involved in isolating a gene, cloning and characterizing it, expressing the encoded protein, purifying the protein, and characterizing rudimentary aspects of its basic physical properties. The manual includes 20 experiments designed to train students to prepare, manipulate, and analyse plasmids, to produce fusion proteins in bacteria, and to purify these proteins based on unique chemical properties or substrate affinities. Electrophoresis, Southern and Western blotting and cominatorial techniques are emphasized. Radioisotopes, fluorophores and solvent effects on protein structure are discussed. (No radioisotopes are required.) The experiments included have been selected because of their high success rate and because they emphasize a project-oriented approach. Sufficient detail and background are provided such that the intended audience includes advanced undergraduate students, beginning graduates, and practising professionals engaged in a broad range of pursuits. Each experiment is accompanied by detailed process rationale. Where appropriate, alternate methods are also discussed. Maximizing its utility for both studetns and instructors, this unique manual precedes each lab experiment with the necessary background theory and principles, which culminate in a detailed process rationale and, ultimately, the experimental techniques themselves. While many lab manuals list background references, this innovative book combines both theory and procedure into one manageable volume. In addition to this theoretical background material, new innovations and insights are provided along with carefully selected primary source research literature.

Introductory Unit, Introductory Lecture - Introduction to the Biochemical Laboratory, Introductory Lab 1 - Basic Biochemical Techniques. Part 1: Pipette Calibration and Solution Preparation. Introductory Lab 2 - Basic Techniques. Part 2: Absorbance Spectroscopy and Protein Concentration Determinations. Part. Part 1:- Nculeic Acids & Cloning, Unit 1, Lecutre 1 - DNA Isolation, Lab 1.1 - Media preparation, Bact, Lab 1.2 - Agarose Gel Electrophoresis, Unit 2, Lecuture 2 - Construction of Recombinant Plasmids, Lab 2.1 - Extraction and Cleanup of DNA Bands from Agrose Gels, Quantification of Yields and Ligation of myo-3 Hind lll DNA Insert Fragment into Linearized ?-gal Plasmic DNA, Unit 3, Lecture 3 - The Polymerase Chain Reactin, 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, Unit 5, Lecture 5 - Transformation and Gene Expression, Lab 5.1 - Preparation of Fresh Transformation - Competent Cells, Lab 5.2 - Colony Immunoblotting to Screen for Transformants, Unit 6, Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and Probing, Lab 6.1 - Labeling of DNA and Probe Construction from Cloned C. elegans myo-3 Gene, Quantitation of DNA Concentration, Lab 6.2 - Isolation of C. elegans Genomic DNA, Quantation of DNA Concetration, and Digestion to Extract the myo-3 Gene, Lab 6.3 - Southern Blotting, Part 2 - Protein Purification, Unit 7, Lecture 7 - Protein Purification, 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, Lab 7.4 - Mciroplate B-Glactosidase Assay to Determine Fractions Containing Fusion Protein, MW Determination, Lab 7.5 - Ion Exchange Column Chromatography, Lab 7.6 - Affinity Chromatography and Microplate b-Glactosidase Assays to Construct an Enzyme Purification Table, Unit 8, Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size Determination, Lab 8.1 - Discontinuous SDS Gel Electrophoresis, Unit 9, Lecture 9 - Immunochemical Techniques, Lab 9.1 - Western Blotting, Unit 10, Lecture 10 - Combinatorial Biochemical Technology, Appendices, Index