Cloning, Gene Expression and Protein Purification
Experimental Procedures and Process Rationale
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Product details:
- Publisher OUP USA
- Date of Publication 17 May 2001
- ISBN 9780195132946
- Binding Paperback
- No. of pages448 pages
- Size 278x217x21 mm
- Weight 1034 g
- Language English
- Illustrations numerous tables 0
Categories
Short description:
This combined lecture and laboratory manual presents 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. Maximizing its utility for both students and instructors, this unique manual precedes each lab experiment with the necessary background theory and principles, which culminate in detailed process rationale followed by the actual experimental technique. While many lab manuals list background references, this innovative book combines both theory and procedure into one manageable volume.
MoreLong description:
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.
Table of Contents:
Introductory Unit
Introductory Lecture - Introduction to the Biochemical Laboratory
Introductory Lab 1 - Basic Biochemical Techniques I: Pipette Calibration and Solution Preparation
Introductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration Determinations
Part I - Nculeic Acids & Cloning
Unit 1
Lecture 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
