Rev. of: Manipulation and expression of recombinant DNA / Susan Carson, Dominique Robertson. 2nd ed. c2006

Includes bibliographical references and index.

Lab Session 1 Getting Oriented: Practicing with Micropipettes
Lab Session 2 Purification and Digestion of Plasmid (Vector) DNA
Lab Session 3 PCR Amplification of egfp and Completion of Vector Preparation
Lab Session 4 Preparation of Insert DNA (egfp) PCR Product
Lab Session 5 DNA Ligation and Transformation of Escherichia coli
Lab Session 6 Colony Hybridization
Lab Session 7 Characterization of Recombinant Clones: Part 1
Lab Session 8 Characterization of Recombinant Clones: Part 2
Lab Session 9 Characterization of Recombinant Clones: Part 3
Lab Session 10 Computational Analysis of DNA Sequence from a Positive Clone: Part 2
Lab Session 11 Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1
Lab Session 12 Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 2
Lab Session 13 Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column
Lab Session 14 Analysis of Purification Fractions
Lab Session 15 Total RNA Purification
Lab Session 16 Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 1
Lab Session 17 Analysis of gst::egfp mRNA Levels by RT-qPCR: Part 2
Lab Session 18 Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 1
Lab Session 19 Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR: Part 2
Appendix 1 Equipment
Appendix 2 Prep List
Appendix 3 Preparation of Competent E. coli Cells
Appendix 4 Pre-Lab Questions

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. *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