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Manipulation and Expression of Recombinant DNA,

Edition 2

Editors: By Sue Carson and Dominique Robertson

Publication Date: 06 Dec 2005

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This manual is an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology, or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students gain hands-on experience from start to finish in subcloning a gene into an expression vector, through purification of the recombinant protein.The second edition has been completely re-written, with new laboratory exercises and all new illustrations and text, designed for a typical 15-week semester, rather than a 4-week intensive course. The “project¿ approach to experiments was maintained: students still follow a cloning project through to completion, culminating in the purification of recombinant protein. It takes advantage of the enhanced green fluorescent protein—students can actually visualize positive clones following IPTG induction.

Key Features

*Cover basic concepts and techniques used in molecular biology research labs*Student-tested labs proven successful in a real classroom laboratories*Exercises simulate a cloning project that would be performed in a real research lab*"Project" approach to experiments gives students an overview of the entire process*Prep-list appendix contains necessary recipes and catalog numbers, providing staff with detailed instructions

About the author

By Sue Carson, North Carolina State University, Raleigh, U.S.A. and Dominique Robertson, North Carolina State University, Raleigh, U.S.A.

CONTENTSForewordAcknowledgementsNote to InstructorsInstrumentationNomenclatureINTRODUCTION: Conceptual outline for experimentsPART I MANIPULATION OF DNALab session 1: Getting oriented; Practicing with pipetmenLab session 2: Large scale purification of plasmid DNA Lab session 3: Preparation of expression vector DNA (pET-41a(+), a GST fusion protein vector)Lab session 4: Preparation of insert DNA (egfp)Lab session 5: Preparation of transformation-competent cells and control transformationLab session 6: DNA ligation and transformation of Escherichia coli PART II SCREENING TRANSFORMANTSLab session 7: Colony hybridizationsLab session 7a: Interim laboratory session:Lab session 7b: Colony hybridization: DNA probeLab session 7c: Colony hybridization: Monoclonal antibody probeLab session 8: Completion of colony hybridization with DNA probeLab session 9: Characterization of recombinant clones Lab session 9a: Completion of colony hybridization with mAB probeLab session 9b: PCR screenLab session 9c: Visualization of green fluorescent protein: Part 1 Lab session 10: Further characterization of recombinant clonesLab session 10a: Interim laboratory session:Lab session 10b: Analysis of PCR screen resultsLab session 10c: Isolation and characterization of miniprep DNA from potential transformants (Restriction analysis of putative transformants)Lab session 10d: Visualization of green fluorescent protein: Part 2 PART III EXPRESSION, DETECTION, AND PURIFICATION OF RECOMBINANT PROTEINS FROM BACTERIALab session 11: Expression of fusion protein from positive clones and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Immunological analysis (Western blot): Part 1Lab session 12: Expression of fusion protein from positive clones and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Immunological analysis (Western blot): Part 2Lab session 13: Extraction of recombinant protein from Escherichia coli using a glutathione affinity columnInterim laboratory session: I. Laboratory exercise: Inoculate cultures for protein purificationLab session 14: Analysis of purification fractionsAppendices: Equipment, Prep list, Making sense of orientation

ISBN: 9780120884186

Page Count: 176

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Graduate and undergraduate students studying biochemistry, molecular biology, biotechnology, and cell biology