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
Description
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.
Readership
Graduate and undergraduate students studying biochemistry, molecular biology, biotechnology, and cell biology
Molecular Biology Techniques, 3rd Edition
CONTENTS
Foreword
Acknowledgements
Note to Instructors
Instrumentation
Nomenclature
INTRODUCTION: Conceptual outline for experiments
PART I 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
PART II 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: Part 2
PART III 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
