2024 Wadsworth
2024 Wadsworth kdorfman Tue, 01/16/2024 - 21:39UNIT 1
| Lab | Day | date | materials |
|---|---|---|---|
| 1 | Th | 2/1 | 1 slide each scope - DAPI |
| 2 | Tu | 2/6 | Same slide as Lab 1, micrometer |
| 3 | Th | 2/8 | 1 slide each scope, DAPI + 1 |
| 4 | Tu | 2/13 | Slides with DAPI, tubulin, actin, also beads for microscope resolution |
| 5 | Th | 2/15 | camera resolution; slides from Lab 4 |
| 6 | Tu | 2/20 | photobleaching. Fresh 3-color slide |
| Th | 2/22 | NO CLASS - MONDAY SCHEDULE | |
| Tu | 2/27 | Presentations |
UNIT 2
| Lab | Day | date | materials |
|---|---|---|---|
| 1 | Th | 2/29 | Immunofluorescence |
| paraglut fixed coverslips1 for tubulin, phalloidin (2 each) | |||
| materials for mounting, slide boxes | |||
| live cells on coverslip for paraglut fix | |||
| 2 | Tu | 3/5 | live cells (GFP-tubulin LL's) in MatTek dishes mitotracker red ER tracker red NBD ceramide |
| 3 | Th | 3/7 | miscellaneous. grow some GFP E. coli |
| 4 | Tu | 3/12 | learn cell culture: 8 12.5 cm2 flasks of HeLa parentals |
| catch-up day: | |||
| 12 dishes live LL-GFP-tub for ER tracker | |||
| 2 fixed coverslips for phalloidin staining | |||
| student made slides from previous lab | |||
| previously made instructor slides |
UNIT 3
| Lab | Day | date | materials |
|---|---|---|---|
| 1 | Tu | 3/26 | 9 coverslips HeLa, 6 coverslips HeLa treated with STLC 5 uM 18 hours, stained for alpha (2ogoat-anti-rat FITC>) and gamma (2ogoat-anti-mouse TRITC) tubulin |
| 2 | Th | 3/28 | HeLa: 3 coverslips, 1 flask, 3 dishes |
| LLCPk-1 GFP alpha 10-15 dishes | |||
| taxol | |||
| nocodazole | |||
| GSK923295 (cenp inhibitor) | |||
| cyto-D | |||
| RO3306 (CDK-1 inhibitor) | |||
| 3 | Tu | 4/2 | HeLa coverslips; LLCPk alpha GFP dishes |
| 4 | Th | 4/4 | HeLa: 2 live, 2 coverslip; LLCPk alpha GFP: 10 live, 4 coverslip |
| 5 | Tu | 4/9 | HeLa: 3 live, 5 coverslips; LLCPk-1: 10 live, 3 coverslips |
| 6 | Th | 4/11 | HeLa: 5 coverslips, 2 dishes; LLCPk-1: 10 live |
| 7 | Tu. | 4/16 | HeLa: 4 coverslips; LLCPk-1: 10 live |
Unit 4
For first day Tu, 4/23: 12 HeLa coverslips
- fixed in methanol
- stained with rat anti alpha tubulin, goat-anti-rat FITC), and 3 stained with each of:
| antibody | final dilution |
|---|---|
| rabbit anti Kif2a | 1:1000 |
| rabbit anti-CAMSAP-2 | 1:500 |
| rabbit anti-CAMSAP-1,2,3 | 1:100 |
| mouse anti-gamma tubulin | 1:20 |
with goat anti-rabbit red as the secondary
Plasmids for Unit 4 from Addgene
| Gene | Addgene # | bacterial selection | mammalian selection |
|---|---|---|---|
| Kif2a-EGFP | 52401 | Kan | Neomycin (G418) |
| CAMSAP-3 neon green | 191322 | amp | |
| EGFP-alpha tubulin | 12298 | amp | Neomycin (G418)/Kan |
| Mcherry tubulin | 31930 | Kan | Neomycin (G418) |
| mCherry H2b | 20972 | Kan (amp) | Neomycin (G418) |
Project 2
| Project | grp | gene | Thu 4/25 | Tu 4/30 | W 5/1 | Th 5/2 |
|---|---|---|---|---|---|---|
| Stable cell line | 1 | Kif2A | mini prep & Nanodrop (Tom) | nucleofect need 100K cells/flask | observe dish, select flask | |
| 6 | Kif2A | |||||
| 8 | Camsap 3 | |||||
| RNAi | 2 | Camsap2 | HeLa live (KD), HeLa mCherry (Pat) | 6 well plates (200 - 250K cells) | coverslips | Fix & Stain |
| 3 | spastin, katanin | |||||
| 4 | camsap3 | |||||
| 5 | spastin | |||||
| 7 | Kif2A |
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HeLa or LLCPk1 ↩︎