| Z700592 Sigma |
Cloning, Gene Expression, and Protein Purification: Experimental Procedures and Process Rationale |
General description
This resource presents protocols for isolating a gene, cloning and characterizing it, expressing its encoded protein, and purifying and characterizing the protein's physical properties. It includes background and procedures and is structured around 20 experiments that demonstrate how to prepare, manipulate, and analyze plasmids, produce fusion proteins in bacteria, and purify these proteins based on chemical properties or substrate affinities. It describes topics such as the use of antibodies and techniques developed to transform their structures, and approaches designed to manipulate structure and functions of proteins and nucleic acids.
Table of Contents
Introductory Unit Introductory Lecture - Introduction to the Biochemical Laboratory
Introductory Lab 1 - Basic Biochemical Techniques I: Pipet Calibration and Solution Preparation
Introductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration Determinations
Part I - Nucleic Acids & Cloning
Unit 1 Lecture 1 - DNA Isolation T
Lab 1.1 - Media Preparation; Bacterial Growths; Plasmid Minipreps; HindIII Digestion of DNA, Commercial Bacteriophage h DNA BstEII Digest Size Standards
Lab 1.2 - Agarose Gel Electrophoresis
Unit 2
Lecture 2 - Construction of Recombinant Plasmids
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
Unit 3 Lecture 3 - The Polymerase Chain Reaction
Unit 4 Lecture 4 - Transcription of Genomic DNA & Analysis of the Resulting mRNAs Alt. A
Unit 5 Lecture 5 - Transformation and Gene Expression
Unit 6 Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and Probing
Part 2 - Protein Purification
Unit 7 Lecture 7 - Protein Purification Theory: Preparation and Handling of Biological Macromolecules for Crystallization Te
Unit 8 Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size Determination
Unit 9 Lecture 9 - Immunochemical Techniques
Unit 10 Lecture 10 - Combinatorial Biochemical Technology
Appendices Part 1 Terms List
Part 2 Terms List Laboratory Reagents Abbreviations
Index
Introductory Lab 1 - Basic Biochemical Techniques I: Pipet Calibration and Solution Preparation
Introductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration Determinations
Part I - Nucleic Acids & Cloning
Unit 1 Lecture 1 - DNA Isolation T
Lab 1.1 - Media Preparation; Bacterial Growths; Plasmid Minipreps; HindIII Digestion of DNA, Commercial Bacteriophage h DNA BstEII Digest Size Standards
Lab 1.2 - Agarose Gel Electrophoresis
Unit 2
Lecture 2 - Construction of Recombinant Plasmids
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
Unit 3 Lecture 3 - The Polymerase Chain Reaction
Unit 4 Lecture 4 - Transcription of Genomic DNA & Analysis of the Resulting mRNAs Alt. A
Unit 5 Lecture 5 - Transformation and Gene Expression
Unit 6 Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and Probing
Part 2 - Protein Purification
Unit 7 Lecture 7 - Protein Purification Theory: Preparation and Handling of Biological Macromolecules for Crystallization Te
Unit 8 Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size Determination
Unit 9 Lecture 9 - Immunochemical Techniques
Unit 10 Lecture 10 - Combinatorial Biochemical Technology
Appendices Part 1 Terms List
Part 2 Terms List Laboratory Reagents Abbreviations
Index
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