Lab Syllabus, Biochemistry: Metabolomics and Regulation Bio 147, 2007
Weds, Jan 17 Lab starts in SW111 for an overview of yeast diauxie and its regulation, the subject of our major laboratory sequence, then we go to the computer center for a briefing about web page design apropos of the ‘yeast as a model for human genetic diseases’ class project.
Weds, Jan 24 Make flow charts of major experiment and minor experiments for lab notebook; growth curves for wild type and YFPSch9 and msn2msn4deletion strains; collect samples for microarray analysis later. Introduction to microarray analysis techniques. Installation of GeneSpring on your computers; examination of data from previous year’s experiments to see trends to look for this year.
Weds Jan 31 Gel filtration minor laboratory. Read in BTS chapter 69, p69 on gel filtration methods. See laboratory notebook pages 2-1 et seq for sample and directions.
RNA preparation for major laboratory sequence. See laboratory notebook pages 1-1 et seq for directions; samples are cells frozen during growth curve last week. Discuss possible predictions of microarray experiments; continue GeneSpring analysis of past data to detect clusters that predict certain pathway changes.
Feb 7. Quality control for RNA samples and planning of microarrays for major experiment sequence (see under laboratory notebook section 1). Completion of analysis of gel filtration column data and discuss in lab; take notes for lab notebook.
Feb 14. Major experiment sequence: Enzyme assays for saved supernatants from growth curve: what happens to the glucose concentration in media during growth? Also, synthesis of the first strand of cDNA from selected mRNA preparations (see under laboratory notebook section 1). Download data from DeRisi experiment on yeast diauxie and examine along with DeRisi paper (class handout), using GeneSpring.
Feb 21. Major experiment sequence: Continue/confirm enzyme assays, discuss glucose in media and write up for lab notebooks. Synthesize second strand of cDNA (see under laboratory notebook section 1).
Feb 28. Major experiment sequence: Prepare amplified RNA from double stranded cDNA. Minor experiment: Inject sample into liquid chromatography ion exchange column and collect data. To prepare, read ion exchange chromatograpy, pp 69-70 in BTS and look at laboratory 3-1 et seq in laboratory notebook. Discuss and take notes in lab book about basis of separation.
March 7. Major experiment sequence: Couple dye to amplified RNA probe and hybridize with microarrays as planned on Feb 7. Arrays washed Feb 8 and scanned in Axon GenePix 2000. Hand in lab notebooks for grading of two column chromatography minor experiments and the preparations for completion of major experiment.
March 14. no laboratory, spring recess
March 21. Major experiment data analysis laboratory 1. Discuss gridding in Magic Tool and in GenePix; grid data and collect intensities. Print out quality control report and put in notebook. Write discussion of microarray quality in lab notebook. Discuss installation of BRB ArrayTools on your computer. Minor experiment: prepare mouse liver and L cell RNA, run QC gel and obtain A260 and A 280 (see section 5-1 et seq in lab notebook). Discuss oncogenes and tumor suppressor genes in mouse.
March 28. Major experiment data analysis laboratory 2. Discussion of statistical issues in microarray analysis. ANOVA using GeneSpring; various data slices using BRB ArrayTools (see section 4-1 et seq in lab notebook). Minor experiment: if mouse RNA is of good quality, prepare cDNA first strand and second strand (section 5 in lab notebook).
April 4. Major experiment data analysis laboratory 3. Presentations by students on findings from microarrays; take notes for your lab notebook about findings, uncertainties, similarities and differences from published data (DeRisi, others you may have found). Minor experiment: prepare aRNA from mouse cDNA (section 5). In AM, someone needs to treat aRNA with DNase for 30 minutes (talk with instructor).
April 11. Major experiment: continue with any work you have decided would clarify your findings. Otherwise, read literature and add insights to your notes under sections 1 and 4. Minor experiment: label and hybridize mouse aRNA to arrays. April 12, wash arrays and scan.
April 18. Minor experiment on mouse gene expression data analysis. At 4-5, talk by John Aris; plan work so you can attend, please.
April 25. Minor experiment on mouse gene expression data analysis, continued. Class discussion of findings comparing L cells and mouse liver.
May 2. Laboratory cleanup and check out. Hand in laboratory notebooks for grading of completion of major experiment and mouse RNA minor experiment. If rewrites were recommended in first grading, they will be regarded at this point.