Caldwell / Williams Integrated Genomics

A Discovery-Based Laboratory Course
1. Auflage 2006
ISBN: 978-0-470-09503-4
Verlag: John Wiley & Sons
Format: PDF
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 246 Seiten, E-Book

Integrated Genomics: A Discovery-Based LaboratoryCourse introduces the excitement of discovery to thebasic molecular biology laboratory. Utilizing up-to-date molecularbiology protocols and a basic experimental design, this text offersexperience with three different model systems. Students will becomefamiliar with the simplicity and power of single-celled organisms,Escherichia coli and Saccharomyces cerevisiae, asthey search for genes that interact and function within thenematode Caenorhabditis elegans.
Incorporated throughout the course are exercises designed tooffer students familiarity with the wealth of bioinformatics datathat can be accessed on the World Wide Web. Following completion ofinteraction studies within the yeast, the course is designed toallow students to examine the functional consequences of reducing agene's function within the multicellular worm that is bothsimple and inexpensive to maintain within a laboratory. Theinclusion of alternative experiments allow for flexibility indetermining the ending date or goal of the laboratory, as well asworking within the available budget and resources of most anyclassroom environment.
Further striking features of this title are:
* An accompanying Web site providing PowerPoint slides, pluslinks to the internet, and regular updates as bioinformaticsdatabases evolve and methods improve. www.wiley.com/go/caldwell
* Inclusion of modern genomic/proteomic technologies such as theyeast two-hybrid system and RNAi
* Detailed experimental protocols and easy access toinstructional materials
This discovery-based laboratory course provides excellentpractical training for those pursuing career paths in biomedicine,pharmacy, and biotechnology.

Autoren/Hrsg.

Caldwell, Guy

Caldwell, Guy A.

Williams, Shelli

Williams, Shelli N.

Caldwell, Kim

Caldwell, Kim A.

Inhaltsverzeichnis

Preface.
Author biographies.
Acknowledgments.
List of figures.
1 Introduction to basic laboratory genetics.
1.1 Transferring and handling C. elegans.
1.2 Introduction to laboratory genetics.
2 Gene expression analysis using transgenic animals.
2.1 Transgenic gene expression analysis in C. elegans: lacZ staining.
2.2 Transgenic gene expression analysis in C. elegans: GFP analysis.
3 Creation and testing of transgenic yeast for use in protein-protein interaction screening.
3.1 Small-scale transformation of S. cerevisiae.
3.2 Transformation of S. cerevisiae to test for non-specific interaction.
3.3 Assaying for protein-protein interaction by reporter gene expression.
4 Yeast two-hybrid screening.
4.1 Protein-protein interaction screening of a C. elegans cDNA library.
4.2 Assaying for protein-protein interaction by reporter gene expression.
5 Isolation and identification of interacting proteins.
5.1 Preparation of electrocompetent E. coli.
5.2 Isolation of DNA from yeast and electroporation of E. coli.
5.3 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.
5.4 Sequencing of two-hybrid library plasmid DNA vectors.
6 Using bioinformatics in modern science.
6.1 DNA sequence chromatogram.
6.2 BLASTing your sequence.
6.3 Evaluating sequence results and choosing an RNAi target.
6.4 Bioinformatics practice questions.
7 Generation of an RNAi vector.
7.1 Small-scale isolation of genomic DNA from C. elegans.
7.2 PCR amplification of target gene sequence from C. elegans genomic DNA.
7.3 Preparations for cloning to generate RNAi vector.
7.3.1 Agarose gel electrophoresis.
7.3.2 Removal of dNTPs from PCR reaction.
7.3.3 Restriction enzyme digestion of PCR product and C. elegans RNAi vector.
7.4 Gel purification of DNA and ligation of vector and PCR-amplified DNA.
7.4.1 Preparative agarose gel electrophoresis.
7.4.2 Gel purification of DNA from agarose gel.
7.4.3 Ligation of vector and PCR-amplified DNA.
7.5 Transformation of ligation reactions.
7.6 PCR screening of transformation colonies.
7.7 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.
7.8 Verifying successful ligation by restriction digestion.
8 RNA-mediated interference by bacterial feeding.
8.1 Preparation of RNAi-feeding bacteria for transformation.
8.2 Media preparation for RNAi feeding.
8.3 Transformation of RNAi-feeding strain HT115(DE3).
8.4 RNA interference by bacterial feeding of C. elegans.
8.5 Analyzing effects of dsRNAi.
8.5.1 Assaying for sterility (Ste) or embryonic lethality (Emb).
8.5.2 Assaying for growth effect.
8.5.3 Assaying for morphological effects.
8.5.4 Assaying for general neuromuscular effects.
8.5.5 Assaying for specific neuronal effects.
8.5.6 Assaying for dauer formation.
Appendix I Recombinational cloning.
AI.1 Isolation of genomic DNA from C. elegans.
AI.2 PCR amplification of target gene sequence from C. elegans genomic DNA.
AI.3 Agarose gel electrophoresis and clean-up of PCR reaction.
AI.4 Entry vector cloning.
AI.5 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.
AI.6 Destination vector cloning.
AI.7 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.
Appendix II Recipes and media preparation.
Solution recipes.
Media preparation.
Appendix III Sterile techniques and worm protocols.
Sterile techniques.
Worm protocols.
Appendix IV Mutant C. elegans phenotypes.
Appendix V Vector maps.
Subject index.

Über Autor(innen)

Guy A. Caldwell, Ph.D., is an Assistant Professor in theDepartment of Biological Sciences at The University of Alabama inTuscaloosa, where since 1999, he has held an undergraduateprofessorial appointment from the Howard Hughes MedicalInstitute. In 2001, Dr. Caldwell was named a Basil O'ConnorScholar of The March of Dimes Birth Defects Foundation for hisresearch into the molecular basis of childhood birth defects of thebrain. Dr. Caldwell is a recipient of grants from The March ofDimes, National Institutes of Health, the Dystonia Medical ResearchFoundation, American Parkinson's Disease Association,Parkinson's Disease Foundation, and the National ParkinsonFoundation. In January 2003, The Caldwell lab was selected as oneof only 11 worldwide to represent the research goals of The MichaelJ. Fox Foundation for Parkinson's Research in their ProteinDegradation Grant Initiative. For his combined teaching andresearch efforts, Dr. Caldwell was also chosen as the recipient ofa 2003 CAREER award from the National Science Foundation, the mostprestigious honor for young faculty bestowed by that organization.He is the author of 2 editions of a widely adopted textbook inbiotechnology sold worldwide in 3 languages by Harcourt. Hecurrently teaches courses in Molecular Genomics, Neuronal SignalingMechanisms, General Biology, and an acclaimed seminar on thesocietal impact of the Human Genome Project.
Shelli N. Williams, B.Sc., is a doctoral candidate in theDepartment of Biological Sciences at The University of Alabama inTuscaloosa, where she has attended the university as anundergraduate and graduate student since 1997. Following herearly graduation magna cum laude from the university, Ms.Williams began her graduate work in the laboratory of Dr. Guy A.Caldwell. She has experience teaching introductory biologycourses to both major and non-major students and has served asteaching assistant for a senior level discovery-based genomicscourse funded by the Howard Hughes Medical Institute.
Kim A. Caldwell, Ph.D. is an Adjunct Assistant Professorin the Department of Biological Sciences at The University ofAlabama Dr. Caldwell serves as an administrative liaison for a 1.8million dollar grant from the Howard Hughes Medical Institute tothe Department of Biological Sciences at Alabama and is Director ofthe HHMI Rural Science Scholars program at Alabama. Shehas designed and taught courses in General Biology, a seminar onthe societal impact of the Human Genome Project, and course is aentitled "The Language of Research" which she teachesjointly for Howard Hughes Research Interns at both Stillman Collegeand The University of Alabama.