Molecular Biology Techniques
A Classroom Laboratory ManualBy
- Sue Carson, North Carolina State University, Raleigh, U.S.A.
- Heather Miller, North Carolina State University, Raleigh, USA
- D. Scott Witherow, North Carolina State University, Raleigh, USA
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 proteinstudents can actually visualize positive clones following IPTG induction.
Graduate and undergraduate students studying biochemistry, molecular biology, biotechnology, and cell biology
Paperback, 232 Pages
Published: November 2011
Imprint: Academic Press
"Whilst molecular biology has been the focus of course curricula in various bioscience educational programmes, there has been a lack of well-designed laboratory manuals to recommend for the practical sessions of these courses. The third edition of Molecular Biology Techniques is one such excellent classroom laboratory manual. It encompasses experiments for 19 laboratory sessions presented as a semester-long project that gets students involved in a comprehensive experimental story from gene cloning to protein purification. The authors have employed the versatility of the PCR technique in various experiments and have also taken advantage of the enhanced green fluorescent protein in visualising positive clones. A new section involving five laboratory sessions on measuring mRNA levels has been added to this third edition. Overall, this manual represents an invaluable training material on practical molecular biology for undergraduates, graduates, and inexperienced researchers. It could also introduce more experienced researchers to experiments that they have not considered previously."--Science Progress
- 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