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 |