BE/APh 162: Winter 2009SyllabusProfessor: Rob PhillipsTAs: Dave Wu, Damien Soghoian, Arbel Tadmor, Hannah TusonMondays, 2-5 PM & Tuesdays, 7-10 PM in 040 KeckWeek 1 –Size of thingsSession 1- Logistics and microscopy fundamentals• Introduction to the course• Microscopy fundamentals & optics demonstration• Köhler illumination• Oil/water/air objectives (demo) ◦ CCD • How it works • Signal quantization and saturation • Pixel resolution ◦ MicromanagerSession 2– Size of things using microscopy▪ Light/phase/fluorescent microscopy ▪ Good practices ▪ Techniques ▪ Making agarose pads ▪ Calibration of microscope using calibration slides ▪ Light/phase microscopy ▪ E. coli grown overnight in poor vs. rich media (Schaechter et al. observation) ▪ Look at an assortment of live bacteria and Eukaryotes• Fluorescent microscopy ◦ YFP labeled phages attached to E. coli ◦ FM dye stained cyanobacteria membranes ◦ Various pre-stained slides Week 2 – Rate of thingsSession 1– Rate of things using microscopy• Light/phase microscopy◦ Movie of E. coli growth ◦ Movie of yeast growth ◦ Beating of Chlamy cilia◦ Dictyostelium• Fluorescent microscopy ◦ Photobleaching of fluorescent E. coli cells Session 2 – Rate of things using spectrophotometry• Theory ◦ Diauxic growth curve using different sugars ▪ Operons and operon regulation ▪ Order of magnitude estimation ◦ Spectrophotometry ▪ Beer-Lambert law ▪ Demonstration on chlorophyll ▪ OD600 vs. cfus • Experimental◦ Measure diauxic growth curve ▪ 1:3 Glucose:Lactose ▪ 1:3 Glucose:Arabinose ▪ 1:3 Glucose:Sorbitol ▪ 1:3 Glucose:Maltose ◦ Plate cellsWeek 3 & 4 – DNA engineeringSession 1– PCR• Outline experiment • PCR YFP insert ◦ PCR protocol ◦ Proper pipetting techniques ◦ Execute PCR • Restriction digest ◦ What we are digesting and why ◦ How to predict cutting sites (internet/Vector NTI) • Project discussionSession 2 – Restriction digests and gels• PCR purification of insert • Digestion ◦ Insert ◦ Plasmid ◦ Lambda• Gel electrophoresis ◦ Cast gels ◦ Talk about gel electrophoresis ◦ Set up samples for gel ◦ Load gel • PCR purification of digested insert Session 3 – Ligation and transformation• Outline for today • Nanodrop PCR insert• Ligation ◦ Figure out volumes ◦ Set up reaction • PCR purification of ligation product • Transformation • Plating Session 4– Analysis of transformed cells• Inspect transformed cells under microscope • YFP induction as a function of IPTG • YFP induction as a function of looping • YFP induction for cells with mutant operators • Measuring expression from Hernan’s YFP/CFP strains • Novick & Weiner single-cell version using fluorescence (Van Oudenaarden paper) Weeks 5-9 – ProjectsWeek 10 – Project
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