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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 third 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
Graduate and undergraduate students studying biochemistry, molecular biology, biotechnology, and cell biology
About the Authors
Note to Instructors
Part 1. Manipulation of DNA
Lab Session 1. Getting Oriented
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
Part 2. Screening Transformants
Lab Session 6. Colony Hybridization
Lab Session 6A. Interim Laboratory Session
Lab Session 6B. Colony Hybridization: Monoclonal Antibody Probe
Lab Session 7. Characterization of Recombinant Clones
Lab Session 7A. Completion of Colony Hybridization with a Monoclonal Antibody Probe
Lab Session 7B. PCR Screening
Lab Session 7C. Prepare Fresh Replica Plate
Lab Session 8. Characterization of Recombinant Clones
Lab Session 8A. Interim Laboratory Session
Lab Session 8B. Analysis of PCR Screen Results
Lab Session 8C. Isolation of Miniprep DNA from Potential Transformants
Lab Session 8D. Visualization of Green Fluorescent Protein: Part 1
Lab Session 9. Characterization of Recombinant Clones
Lab Session 9A. Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants)
Lab Session 9B. Visualization of Green Fluorescent Protein: Part 2
Lab Session 9C. Computational Analysis of DNA Sequence from a Positive Clone: Part 1
Lab Session 10. Computational Analysis of DNA Sequence from a Positive Clone
Part 3. Expression, Detection and Purification of Recombinant Proteins from Bacteria
Lab Session 11. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot
Lab Session 11A. Interim Laboratory Session
Lab Session 11B. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot
Lab Session 12. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot
Lab Session 13. Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column
Lab Session 13A. Interim Laboratory Session
Lab Session 13B. Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column
Lab Session 14. Analysis of Purification Fractions
Lab Session 14A. Analysis of Purification Fractions
Lab Session 14B. Replica Plate Positive Clone
Part 4. Analysis of mRNA Levels
Lab Session 15. Total RNA Purification
Lab Session 15A. Interim Laboratory Session
Lab Session 15B. Total RNA Purification
Lab Session 16. Analysis of gst::egfp mRNA Levels by RT-qPCR
Lab Session 17. Analysis of gst::egfp mRNA Levels by RT-qPCR
Lab Session 18. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR
Lab Session 19. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR
Appendix 1. Equipment
Appendix 2. Prep List
Appendix 3. Preparation of Competent E. coli Cells
Appendix 4. Pre-Lab Questions
- No. of pages:
- © Academic Press 2012
- 7th November 2011
- Academic Press
- eBook ISBN:
- Paperback ISBN:
Dr. Heather Miller is an Assistant Professor of Biochemistry in the Chemistry Department at High Point University (High Point, NC). She graduated from Clarion University of Pennsylvania (Clarion, PA) with a B.S. in Molecular Biology/Biotechnology, and from Duke University (Durham, NC) with a Ph.D. in Molecular Genetics and Microbiology. She completed a teaching postdoctoral position in the Biotechnology Program at North Carolina State University (Raleigh, NC). Her area of scientific expertise is RNA biology. Her research focuses on HIV-1 gene expression and the coupling of transcription and splicing in humans. She has taught at the college level for nine years, and is engaged in the scholarship of teaching and learning.
Department of Chemistry, High Point University, High Point, NC USA
Dr. Witherow graduated from Rollins College (Winter Park, FL) with an A.B. in Chemistry, and from the University of Miami (Miami, FL) with a Ph.D. in Molecular and Cellular Pharmacology. His research has focused primarily on G protein-mediated signal transduction processes in mammalian systems. Following two research postdoctoral fellowships, Dr. Witherow served as a teaching postdoctoral fellow at North Carolina State University, where he published and presented multiple papers in the field of science education and developed a passion for teaching. He is currently an Associate Professor at The University of Tampa, where he has been teaching undergraduate students since 2011.
Department of Chemistry, Biochemistry, and Physics, The University of Tampa, Tampa, FL USA
North Carolina State University, Raleigh, U.S.A.
"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, 2012
"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
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