Incorporated throughout the course are exercises designed to offer students familiarity with the wealth of bioinformatics data that can be accessed on the World Wide Web. Following completion of interaction studies within the yeast, the course is designed to allow students to examine the functional consequences of reducing a gene’s function within the multicellular worm that is both simple and inexpensive to maintain within a laboratory. The inclusion of alternative experiments allow for flexibility in determining the ending date or goal of the laboratory, as well as working within the available budget and resources of most any classroom environment.
Further striking features of this title are:
- An accompanying Web site providing PowerPoint slides, plus links to the internet, and regular updates as bioinformatics databases evolve and methods improve. [external URL]
- Inclusion of modern genomic/proteomic technologies such as the yeast two-hybrid system and RNAi
- Detailed experimental protocols and easy access to instructional materials
This discovery-based laboratory course provides excellent practical training for those pursuing career paths in biomedicine, pharmacy, and biotechnology.
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.
Appendix III Sterile techniques and worm protocols.
Appendix IV Mutant C. elegans phenotypes.
Appendix V Vector maps.