2025 Maresca

Gene & Genome Analysis (Bio 383)

Weekclassdaydatetopics
11W9/3Tom: Intro to the course
Kate: intro to safetyfluorescence microscope
live GFP-tubulin S2 cell imaging on 8 conA coverslip dishes (prepared just before class)
pipetting part 1
21M9/8pipetting part 2
microscopy HeLa slides
S2 culture (24 flasks – each student makes one)
3W9/10Transformation
34M9/15mini preppcr
5W9/170.9% gel, band extraction
46M9/22invitro transcription kitconA dishes
7W9/24gel, + Nanodrop
58M9/29ds RNA treatment in 2 wells of a 6-well dish
9W10/116 conA dishes (Tom)
5 mL S2 aliquots
610M10/6IF demo (Kate)
Handout
repeat ds RNA treatment
make 2 conA coverslip per group
11W10/8immunostaining
Handout2
Revised immunostaining for S2 cells
IF (students seed coverslips and fix) on control and dsRNA treated cells
7M10/13Indigenous People’s Day
12W10/15looking at coverslips (Ctrl vs treated)
813M10/20work on poster presentation
14W10/22presentations (in ILC)
Get HeLa from Tom, put on coverslips to treat & stain for 10/27
F10/24HeLa 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
915M10/27Tom 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
16W10/29Imaging dishes with nucleofected HeLa from 10/27
Split flasks from 10/27
F10/31Tom makes coverslips from student nucleofected flasks + control
Sun11/3Tom treats coverslips with STLC
1017M11/3Morning: 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)
18W11/5Gibson cloning (part II): miniprep and test digests
transfection of HeLa cells
(PX458GFP)
Miniprep, nanodrop, restriction digest (Bsb1)
1119M11/101st CRISP experiment: Nucleofection of CRISPR construct into HeLa cells
20W11/12Live-cell imaging to look for transfection efficiency
IF
1221M11/17IF to assess knockout efficiency
22W11/19Set up 2nd CRISPR experiment: Nucleofection of different guide RNAs
1323M11/24IF and Knockout efficienty
W11/26Thanksgiving Break
1424M12/1Nocodazole treatment
IF
25W12/3Look at fixed cells, quantify, work on presentation
1526M12/8CRISPR Group Presentation
Extraction numberng/uL DNA
1345
2221
3350
42052
5108
6149
7306
8246
9302
10137
11223
12210