Gene & Genome Analysis (Bio 383)

Pre-semester reagent list from 2022

Revised syllabus 10/31/25

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 stain with Nuc Blue check for transformation efficiency Split flasks from 10/27 (not actually ready to split on 10/29) Kate makes 10 control coverslips, to be treated with STLC, then fixed. Make 250 µL 1 mM STLC from 100 mM stock: (2.5 µL + 250 µL DMSO) Add 10 µL 1mM stock to each 2mL well to make 5 µM STLC
		Th	10/30	Kate: treat HeLa controls with 5 µM STLC at end of day.
		F	10/31	Kate fixes HeLa controls (4% PFA) Tom: makes coverslips from student nucleofected flasks + control
		Sun	11/3	Tom treats coverslips with STLC
10	17	M	11/3	Morning: Kate fixes (4% PFA) coverslips from 11/3 Block by adding 10% BSA 15 min before class Make primaries: tubulin (rat) & Ki67 (rabbit) Make secondaries: Anti-rat green, anti-rabbit red New protocol New protocol for BSC fix and IF knockdown HeLa; image
	18	W	11/5	Fixed cell analysis to look for loss of Ki-67 and for the chromosome phenotype
11	19	M	11/10	Prepare plasmids for Gibson assembly reactions Restriction digest of PX458GFP, 2 per group in pcr tubes, in thermocycler. One uncut plasmid for control (Bbs1) (new from NEB freezer 10/31/25 R3539S) Small is available at NEBNow, need 2 or special order the large. Clone construct, prep vector 1% gel NEB 1Kb marker and gel extraction kit (1 per group – students combine their 2 cut plasmids) Nanodrop Traces are weird – unknown peak at about 245 nm, 260/280 ratios as high as 20!
	20	W	11/12	Gibson assembly (isothermal) reaction with chosen DNA from Twist and PX458GFP *Magic Mix 15 µL: (enzymes from “Maresca” box) • T5 exonuclease • Phusion polymerase • Taq ligase Cut plasmid 50 – 100 ng Insert equimolar amount (we will measure this for you) DNAse-free water up to 20 µL Incubate at 50°C for 15 minutes Transform 5-10 µL of reaction into competent E. coli cells bacterial Transformation
12	21	M	11/17	Restriction digest: EcoR1 pcr tubes for reaction 1% SYBR safe gel 1 KB ladder NEB Minipreps and plasmidsaurus sequencing
	22	W	11/19	DNA sequence analysis and nucleofection of your plasmid into HeLa cells Students make one flask and seed one coverslip
		F	11/21	Kate: Fix coverslips in 4%PFA; store in PBS-Tw-Azide Seed new coverslips from Wed nucleofected flasks
13	23	M	11/24	Before class: Kate: fix Fri coverslips in 4%PFA; block all. Immunofluorescence to assay for KO phenotype of your target gene.
		W	11/26	Thanksgiving Break
14	24	M	12/1	Fixed cell analysis to look for phenotype
	25	W	12/3	Continuing looking at fixed cells, quantify, and group work on poster presentations
15	26	M	12/8	CRISPR Group Presentation

Extraction number	ng/µL 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