Molecular Biology Techniques: A Classroom Laboratory Manual, Fourth Edition, is a must-have collection of methods and procedures that create a single, continuous, comprehensive project that teaches students basic molecular techniques. It 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 fourth edition is updated with new laboratory exercises, designed for a typical 15-week semester, rather than a 4-week intensive course. The "project" approach to experiments has been student and instructor tested for years: 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.
- Student-tested labs proven successful in real classroom laboratories
- Testbank included on companion website for additional testing and practice
- 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
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 2. Prep List 3. Preparation of Competent E. coli Cells 4. Pre-Lab Questions
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.
Srougi, Melissa C.
Dr. Melissa C. Srougi is an Assistant Professor of Biochemistry at High Point University. She graduated from the University of Toledo (Toledo, OH) with a B.S. in Biology, and from Case Western Reserve University (Cleveland, OH) with a Ph.D. in Pharmacology. Her scientific areas of expertise are in experimental cancer chemotherapeutic agents and mechanisms of chemotherapeutic resistance. Dr. Srougi actively trains undergraduate research students in her laboratory. In addition, she has developed and taught a variety of inquiry-based college level science courses and has published and presented a number of peer-reviewed papers in the scholarship of teaching and learning.
Witherow, D. Scott
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