Integrated Genomics: A Discovery-Based Laboratory Course / Edition 1

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Overview

Integrated Genomics: A Discovery-Based Laboratory Course introduces the excitement of discovery to the basic molecular biology laboratory. Utilizing up-to-date molecular biology protocols and a basic experimental design, this text offers experience with three different model systems. Students will become familiar with the simplicity and power of single-celled organisms, E.coli and S.cerevisiae, as they search for genes that interact and function within the nematode C.elegans. They will also begin to explore the wealth of bioinformatics data available on the Internet via numerous exercises incorporated throughout the book.

Using simple and inexpensive techniques, the concluding chapters enable students to examine the phenotypic consequences of reducing gene function within C.elegans. The authors have included a range of alternative experiments that offer flexibility in determining the end date or goal of the laboratory, as well as working within the available budget and resources of most classroom environments.

Key Features: Provides training with multiple inexpensive and popular model organisms including C.elegans and S.cerevisiae, Adopts a student-tested, discovery-based format, Integrates basic bioinformatics within an experimental context, Inclusive of modern molecular genomic/proteomic technologies such as the yeast two-hybrid system and RNAi, Accompanying website includes easy access to instructional materials and experimental reagents including all strains and plasmids used in the course. An invaluable text for advanced undergraduate and postgraduate students studying cell and molecular biology, genetics, microbiology, biochesmistry, proteomics, genomics andbioinformatics.

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Editorial Reviews

From the Publisher

"I greatly admire the efforts of the authors. Their goals are praiseworthy and this manual is well written." (The Quarterly Review of Biology, September 2007)

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Product Details

  • ISBN-13: 9780470095010
  • Publisher: Wiley
  • Publication date: 9/25/2006
  • Edition number: 1
  • Pages: 246
  • Product dimensions: 8.80 (w) x 11.12 (h) x 0.81 (d)

Meet the Author

Guy A. Caldwell, PhD, is an Associate Professor in the Department of Biological Sciences at the University of Alabama, where since 1999 he has held an undergraduate professorial appointment from the Howard Hughes Medical Institute. He holds an adjunct appointment at the University of Alabama at Birmingham, as an Assistant research Professor of Neurology. In 2001, Dr Caldwell was named a Bail O' Connor Scholar of The March of Dimes Birth Defects Foundation for his research into the molecular basis of childhood birth defects of the brain. Dr Caldwell is a recipient of grants from The March of Dimes, National Institutes of Health, Dystonia Medical Research Foundation, Parkinson's Disease Foundation, National Parkinson Foundation, and the Bachmann-Strauss Dystonia & Parkinson Foundation. In 2003, The Caldwell Laboratory was selected as 1 0f 11 groups worldwide to represent the research goals of the Michael J. Fox Foundation for Parkinson's research in their Protein Degradation Grant Initiative. For his combines teaching and research efforts, Dr Caldwell was also chosen as the recipient of a 2003 CAREER Award from the National Science Foundation. In 2005, he was named Alabama Professor of the Year by the Carnegie Foundation for the Advancement of Teaching and Council for Advancement and Support of Education. Dr Caldwell, a native of the New York City area, received his undergraduate degree in Biology from Washington & Lee University in 1986 and his PhD in Cell, Molecular & Developmental Biology from The University of Tennessee in 1993. Following receipt of his doctorate, he moved to Columbia University in New York where he was twice named the recipient of fellowships from the National Institute of Neurological Disease and Stroke. He is the author of two editions of a widely adopted textbook, Biotechnology: A Laboratory Course, published worldwide in three languages. He teaches courses in Integrated Genomics, Neuronal Signaling, General biology, and an acclaimed seminar on the societal impact of the Human Genome Project.

Shelli N. Williams, PhD, is a research scientists at a private forensic company based in Virginia. Following her early graduation Magna cum laude from undergraduate studies, Dr Williams began her graduate work in the laboratory of Drs Guy and Kim Caldwell at The University of Alabama, where she earned her doctorate from The University of Alabama in 2006. Dr Williams served as an adjunct faculty member in New College, an interdisciplinary department at The University of Alabama, where she was the instructor of a seminar course demonstrating how the nature of the laboratory experience plays an essential role in the understanding and advancement of science. She has experience plays an an essential role in the understanding and advancement of science. She has experience teaching introductory biology courses to both majors and non-majors students and has been a repeated guest lecturer in a cross-disciplinary bioethics class. As a PhD candidate, Dr Williams served as a teaching assistant for Integrated genomics, a discovery-based genomics course funded by the Howard Hughes Medical Institute. Dr Williams was named the recipient of two university-wide Graduate Council Fellowships, as well as receiving recognition as an Isabella Hummel graham Scholar honoring outstanding female students throughout the university. She also received a competitive Worthington Biochemical Travel Award from the American Society of Cell Biology, Placing her among the highest honored student researchers at their 2003 Conference. Subsequent graduate work establishing Caenorhabditis elegans as a model for epilepsy was high-lighted in news releases by the Howard Hughes Medical Institute. In recognition of her accomplishments, Dr Williams was awarded the 2005 Joab Langston Thomas Award, the top honor for PhD students in Biological Sciences at the University of Alabama.

Kim A. Caldwell, PhD, is an Assistant Professor in the Department of Biological Sciences at The University of Alabama. Dr Caldwell is a Faculty Affiliate of The University of Alabama Center for Green Manufacturing and she is an Adjunct research Assistant Professor in the Department of Neurology at the University of Alabama at Birmingham Medical School. Dr Caldwell, a native of the Buffalo area, received her undergraduate degree in recombinant Gene Technology from the State University of New York at Fredonia and her MS and PhD degrees in Biotechnology and Cell, Molecular & Developmental Biology, respectively, from The University of Tennessee, While at Tennessee, Dr Caldwell was a four-time recipient of the Oak Ridge National Lab-UT Science Alliance teaching/research Award and the Chancellor's Award for Extraordinary Professional Promise. Following receipt of her doctorate, she held postdoctoral research appointments at the Rockefeller University and Columbia University in New York, during which time he was named the recipient of a Revson Fellowship and a National research Service Award from the National Institue of Child Health and Human Development. Her research has been published in many outstanding peer-reviewed journals, including Nature, Proceedings of the National Academy of Sciences, Journal of Neuroscience, Human Molecular Genetics the Journal of Cell Science and Development. Dr Caldwell serves as Director of the Howard Hughes Medical Institute rural Science Scholars program at Alabama. Additionally, she has designed and taught courses in General Biology, a seminar on the societal impact of the Human Genome Project, and a course entitled ' the Language of research', which she teaches jointly for Howard Hughes research Interns at both Stillman College and the University of Alabama. For her teaching efforts,in 2005 Dr Caldwell was selected as a Education Fellow in the Life Sciences of the National Academy of Sciences.

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Table of Contents


Preface     xi
Author biographies     xiii
Acknowledgments     xvii
List of figures     xix
Introduction to basic laboratory genetics     1
Transferring and handling C. elegans     5
Introduction to laboratory genetics     8
Gene expression analysis using transgenic animals     15
Transgenic gene expression analysis in C. elegans: lacZ staining     17
Transgenic gene expression analysis in C. elegans: GFP analysis     22
Creation and testing of transgenic yeast for use in protein-protein interaction screening     27
Small-scale transformation of S. cerevisiae     31
Transformation of S. cerevisiae to test for non-specific interaction     36
Assaying for protein-protein interaction by reporter gene expression     42
Yeast two-hybrid screening     47
Protein-protein interaction screening of a C. elegans cDNA library     47
Assaying for protein-protein interaction by reporter gene expression     54
Isolation and identification of interacting proteins     61
Preparation of electrocompetent E. coli     61
Isolation of DNA from yeast and electroporation of E. coli     65
Small-scale isolation of plasmid DNA from E. coli: the mini-prep     71
Sequencing of two-hybrid library plasmid DNA vectors     76
Using bioinformatics in modern science     83
DNA sequence chromatogram     84
BLASTing your sequence     85
Evaluating sequence results and choosing an RNAi target     86
Bioinformatics practice questions     89
Generation of an RNAi vector     93
Small-scale isolation of genomic DNA from C. elegans     93
PCR amplification of target gene sequence from C. elegans genomic DNA     97
Preparations for cloning to generate RNAi vector     105
Agarose gel electrophoresis     107
Removal of dNTPs from PCR reaction     110
Restriction enzyme digestion of PCR product and C. elegans RNAi vector     111
Gel purification of DNA and ligation of vector and PCR-amplified DNA     117
Preparative agarose gel electrophoresis     118
Gel purification of DNA from agarose gel     119
Ligation of vector and PCR-amplified DNA     121
Transformation of ligation reactions     122
PCR screening of transformation colonies     126
Small-scale isolation of plasmid DNA from E. coli: the mini-prep     130
Verifying successful ligation by restriction digestion      133
RNA-mediated interference by bacterial feeding     137
Preparation of RNAi-feeding bacteria for transformation     139
Media preparation for RNAi feeding     143
Transformation of RNAi-feeding strain HT115(DE3)     147
RNA interference by bacterial feeding of C. elegans     150
Analyzing effects of dsRNAi     156
Assaying for sterility (Ste) or embryonic lethality (Emb)     158
Assaying for growth effect     159
Assaying for morphological effects     159
Assaying for general neuromuscular effects     161
Assaying for specific neuronal effects     163
Assaying for dauer formation     165
Recombinational cloning     169
Isolation of genomic DNA from C. elegans     170
PCR amplification of target gene sequence from C. elegans genomic DNA     170
Agarose gel electrophoresis and clean-up of PCR reaction     171
Entry vector cloning     171
Small-scale isolation of plasmid DNA from E. coli: the mini-prep     173
Destination vector cloning     174
Small-scale isolation of plasmid DNA from E. coli: the mini-prep     176
Recipes and media preparation     177
Solution recipes     177
Media preparation     182
Sterile techniques and worm protocols     197
Sterile techniques     197
Worm protocols     199
Mutant C. elegans phenotypes     205
Vector maps     215
Subject index     219
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