Molecular biology techniques: a classroom laboratory manual (3rd Ed.) CARSON Sue, MILLER Heather, WITHEROW D. Scott

Lavoisier S.A.S. 14 rue de Provigny 94236 Cachan cedex FRANCE Heures d'ouverture 08h30-12h30/13h30-17h30 Tél.: +33 (0)1 47 40 67 00 Fax: +33 (0)1 47 40 67 02 Url canonique : Url courte ou permalien : Url canonique : 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. INTRODUCTION: Conceptual outline for experiments. MANIPULATION OF DNA. Lab session 1: Getting oriented, Practicing with pipetmen. Lab 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 transformation. Lab session 6: DNA ligation and transformation of Escherichia coli. SCREENING TRANSFORMANTS. Lab session 7: Colony hybridizations. Lab session 7a: Interim laboratory session: Lab session 7b: Colony hybridization: DNA probe. Lab session 7c: Colony hybridization: Monoclonal antibody probe. Lab session 8: Completion of colony hybridization with DNA probe. Lab session 9: Characterization of recombinant clones. Lab session 9a: Completion of colony hybridization with mAB probe. Lab session 9b: PCR screen. Lab session 9c: Visualization of green fluorescent protein: Part 1 . Lab session 10: Further characterization of recombinant clones. Lab session 10a: Interim laboratory session:. Lab session 10b: Analysis of PCR screen results. Lab session 10c: Isolation and characterization of miniprep DNA from potential transformants (Restriction analysis of putative transformants). Lab session 10d: Visualization of green fluorescent protein. EXPRESSION, DETECTION, AND PURIFICATION OF RECOMBINANT PROTEINS FROM BACTERIA. Lab session 11: Expression of fusion protein from positive clones and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Immunological analysis (Western blot): Part 1. Lab session 12: Expression of fusion protein from positive clones and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Immunological analysis (Western blot): Part 2. Lab session 13: Extraction of recombinant protein from Escherichia coli using a glutathione affinity column. Interim laboratory session: I. Laboratory exercise: Inoculate cultures for protein purification. Lab session 14: Analysis of purification fractions. Appendices: Equipment, Prep list, Making sense of orientation. Source.

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