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)

"…an excellent tool for introducing a wide range of students to modern concepts in molecular biology, genomics, proteomics, and bioinformatics in an integrated discovery-based format…" (The Annals of Pharmacotherapy, March 2007)

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Product Details

  • ISBN-13: 9780470095027
  • Publisher: Wiley
  • Publication date: 9/26/2006
  • Edition description: New Edition
  • Edition number: 1
  • Pages: 248
  • Product dimensions: 7.44 (w) x 9.69 (h) x 0.63 (d)

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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