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Molecular Biology Techniques
A Classroom Laboratory Manual
- 3rd Edition - October 18, 2011
- Authors: Heather B. Miller, D. Scott Witherow, Sue Carson
- Language: English
- Paperback ISBN:9 7 8 - 0 - 1 2 - 3 8 5 5 4 4 - 2
- eBook ISBN:9 7 8 - 0 - 1 2 - 3 8 5 5 4 5 - 9
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 ex… Read more
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Request a sales quoteThis 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
Preface
About the Authors
Acknowledgements
Note to Instructors
Instrumentation
Nomenclature
Introduction
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
Index
- No. of pages: 232
- Language: English
- Edition: 3
- Published: October 18, 2011
- Imprint: Academic Press
- Paperback ISBN: 9780123855442
- eBook ISBN: 9780123855459
HM
Heather B. Miller
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.
Affiliations and expertise
Department of Chemistry, High Point University, High Point, NC USADW
D. Scott Witherow
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.
Affiliations and expertise
Department of Chemistry, Biochemistry, and Physics, The University of Tampa, Tampa, FL USASC
Sue Carson
Dr. Carson’s area of scientific expertise is in molecular mechanisms of bacterial pathogenesis. She currently serves as the Director of the Master of Microbial Biotechnology Program and as a Fellow in the Office of Faculty Excellence. Prior to this appointment, she led the NC State Quality Enhancement Plan (QEP), focused on faculty development to cultivate students’ critical and creative thinking skills across disciplines. During that period, she also acted as Executive Director of Academic Enrichment Programs, overseeing the Office of Undergraduate Research, Fellowship Advising, and the University Honors and Scholars Programs. Dr. Carson spent over ten years leading curriculum development for the NC State Biotechnology Program. She has received multiple awards for teaching excellence and innovation and was a member of the Howard Hughes Science Education Alliance, promoting and implementing inquiry-guided learning and authentic research in undergraduate curricula. She authored three molecular biology lab manuals and has published numerous peer-reviewed papers in the area of course and curriculum development. She has mentored over 100 undergraduate students in research projects and was the PI and Director of the National Science Foundation (NSF)-funded Integrative Plant and Microbial Systems Research Experience for Undergraduates (REU) Program for over a decade. Most recently, she completed a 2-year rotation at the National Science Foundation in the Directorate for Education and Human Resources. Within the Triangle community, she has served on the Board of Directors of the Wake County Beekeeping Association and the Triangle Swing Dance Society.
Affiliations and expertise
North Carolina State University, Raleigh, U.S.A.Read Molecular Biology Techniques on ScienceDirect