Synopsis
This is a manual which aims to give students a chance to explore the process and techniques of characterizing and purifying a protein, and of subsequent cloning of the associated gene. Each chapter emphasizes the process of discovery, and the strategies and rationale for each experiment. To minimize cost and avoid extensive laboratory preparation, the experiments in this manual use readily available materials and organisms, the bacterium Escherichia coli and the yeast Saccharomyces carlsbergensis. Laboratory topics include: microbiological methods (handling, storage, media); organism growth and protein production; enzyme and protein essays (specific activity); batch purification of proteins; gel filtration and ion exchange chromatographies; genomic and plasmid DNA isolation; and DNA sequencing and computer analysis.
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