Gene & Genome Analysis (Bio 383)

Week	class	day	date	topics
1	1	W	9/3	Tom: Intro to the course Kate: intro to safety, fluorescence microscope live GFP-tubulin S2 cell imaging on 8 conA coverslip dishes (prepared just before class) pipetting part 1
2	1	M	9/8	pipetting part 2 microscopy HeLa slides S2 culture (24 flasks – each student makes one)
	3	W	9/10	Transformation
3	4	M	9/15	mini prep, pcr
	5	W	9/17	0.9% gel, band extraction
4	6	M	9/22	invitro transcription kit; conA dishes
	7	W	9/24	gel, + Nanodrop
5	8	M	9/29	ds RNA treatment in 2 wells of a 6-well dish
	9	W	10/1	16 conA dishes (Tom) 5 mL S2 aliquots
6	10	M	10/6	IF demo (Kate) Handout repeat ds RNA treatment make 2 conA coverslip per group
	11	W	10/8	immunostaining Handout2 Revised immunostaining for S2 cells IF (students seed coverslips and fix) on control and dsRNA treated cells
7		M	10/13	Indigenous People’s Day
	12	W	10/15	looking at coverslips (Ctrl vs treated)
8	13	M	10/20	work on poster presentation
	14	W	10/22	presentations (in ILC) Get HeLa from Tom, put on coverslips to treat & stain for 10/27
		F	10/24	HeLa into small flasks for students 10/27 HeLa STLC treated coverslips (5 µM overnight), fixed and stained for tubulin (red) & PhosH3 (green) Transform E. coli (NEB C2988) with PX458GFP; miniprep to get plasmid See DNA concentrations below
9	15	M	10/27	Tom mixes DNA with Mirus, aliquots splitting demo: Splitting handout Splitting & nucleofection PowerPoint Nucleofection protocol for sterile hood 1 HeLa flask per group (8) Get DNA from freezer Students trypsinize, put some into flask, the rest into nucleofection. Nucleofected cells into flask, imaging dish. transformations
	16	W	10/29	Imaging dishes with nucleofected HeLa from 10/27 Split flasks from 10/27
		F	10/31	Tom makes coverslips from student nucleofected flasks + control
		Sun	11/3	Tom treats coverslips with STLC
10	17	M	11/3	Morning: Kate fixes coverslips from 11/3 Block with 10% BSA 15 min before class Make primaries: tubulin (rat) & PhosH3 (rabbit) Make secondaries: Anti-rat red, anti-rabbit green fix and IF knockdown HeLa; image (Gibson cloning (part I): gel, gel extraction (1 per student), anneal primers (1 per Group), isothermal reaction, Transformation of Competent Cells 45 min gel rather than 60 min? 15 min, not 15 sec on thermocycler for isothermal rxn split HeLas while gels run)
	18	W	11/5	Gibson cloning (part II): miniprep and test digests transfection of HeLa cells (PX458GFP) Miniprep, nanodrop, restriction digest (Bsb1)
11	19	M	11/10	1st CRISP experiment: Nucleofection of CRISPR construct into HeLa cells
	20	W	11/12	Live-cell imaging to look for transfection efficiency IF
12	21	M	11/17	IF to assess knockout efficiency
	22	W	11/19	Set up 2nd CRISPR experiment: Nucleofection of different guide RNAs
13	23	M	11/24	IF and Knockout efficienty
		W	11/26	Thanksgiving Break
14	24	M	12/1	Nocodazole treatment IF
	25	W	12/3	Look at fixed cells, quantify, work on presentation
15	26	M	12/8	CRISPR Group Presentation

Extraction number	ng/uL DNA
1	345
2	221
3	350
4	2052
5	108
6	149
7	306
8	246
9	302
10	137
11	223
12	210