Molecular Techniques Workshop
Dec. 6, 8, 9, 12, 14, 2011
Objectives
Introduce graduate students to most important molecular tools for research, and introduce them to basic techniques in practical exercises. The aim is to familiarize students with techniques so that they can better understand the modern plant literature, and are aware of techniques that might be applicable to their work.
Organization:
The course is organized into morning lectures (2-3 h) in Cunn 146, followed by afternoon lab exercises, 5 days.
Should register for Biol 550E Directed studies (for 1.0 credits) (see Eleanore).
Evaluation (credit students only, each component valued equally)
1. Mini Lab-Report Written summary of results from the recombinant protein experiments. Please write individually but obviously you can consult. Include the data. If the outcome is unexpected, discuss why (3-4 pp. should be sufficient)
2. Write a short proposal, based on your thesis work (or related side project), illustrating how you might be able to use some of the molecular techniques discussed in your research. (approx. 5 pp. double spaced, but concise). I realize some projects are better suited to this than others, but I will take this into consideration.
Day 1 (Tues Dec 6th) Molecular Cloning. Vectors, Enzymes
AM Lecture - Introduction to cloning: vectors and systems
- key enzymes for molecular biology
- Gateway cloning: systems and applications (K Yoshida)
PM Lab: Ligation and Bacterial transformation
1) Ligation of PCR product
2) heat shock transformation
Day 2 (Thurs Dec 8th) Functional Analysis of Genes
AM Lecture - Overview of expression systems for recombinant proteins
- recombinant proteins
- RNAi and tranformation systems
Begin Lab 11 am: 1) capture of recombinant protein by Ni-resin
PM Lab: Recombinant Protein Analysis Part I
2) Purify recGT by mini-column
3) - set up GT assay
Day 3 (Fri Dec 9th) PCR and Gene Expression
AM Lecture: - Overview of PCR applications, cloning with PCR, mutagenesis
- qPCR other assays of gene expression
- microarrays, RNAseq approaches.
PM Lab: Recombinant Protein Analysis Part II
4) SDS-PAGE and western blots
5) GT Assay: spin, do Strata-X
Day 4 (Mon Dec 12th) Molecular Tools for Microbial Ecology
AM Lecture. R Winder (1-1.5 h? ) Molecular Methods for Soil microbes
- Demo/Tour of Pacific Forestry Centre and Winder lab
PM Lab (UVic): Recombinant Protein Analysis Part III
6) Perform western blots with Antibodies and develop
7) Analyse western blots, calculate MW
8) Analyse HPLC traces, calculate specific activity?
Day 5 (Wed Dec 14th) Analytical Tools and Methods (V Walker/D. Smith)
AM Lecture - Overview of analytical methods (V Walker)
- Demonstration/Lab: Protoplast Isolation (LAB - Lan - about 1-1.5 h)
PM: Proteomics Centre: Lecture and tour by D. Smith (2-3 h)