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

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.

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