Tom Maresca’s first run of G&GA
| class | day | date | topics |
|---|---|---|---|
| 1 | W | 9/7 | Tom: Intro to the course Kate: intro to safety, fluorescence microscope live cell imaging on conA coverslip dishes |
| 2 | M | 9/12 | pipetting, S2 culture, microscopy |
| 3 | W | 9/14 | Transformation |
| 4 | M | 9/19 | mini prep, pcr |
| 5 | W | 9/21 | 0.9% gel, band extraction |
| 6 | M | 9/26 | invitro transcription kit; conA dishes |
| 7 | W | 9/28 | gel, + Nanodrop |
| 8 | M | 10/3 | ds RNA treatment |
| 9 | W | 10/5 | 16 conA dishes (Tom) 5 mL S2 aliquots |
| 10 | W | 10/12 | immunostaining |
| 11 | M | 10/17 | repeat ds RNA treatment make 1 conA coverslip per student |
| 12 | W | 10/19 | IF (students seed coverslips and fix) on control and dsRNA treated cells |
| 13 | M | 10/24 | looking at coverslips (Ctrl vs treated) |
| 14 | W | 10/26 | presentations (in ILC?) |
| 15 | M | 10/31 | transformations (PXGFP); STLC (dishes); splitting demo |
| 16 | W | 11/02 | Miniprep, nanodrop, restriction digest (Bsb1) |
| 17 | M | 11/07 | 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 | 11/09 | mini prep, (skip nanodrop) 1 uL 30 min test digest (BsmB1) 45 min test gel split cells during either digest or gel, depending on lecture |
| 19 | M | 11/14 | transfection of HeLa cells |
| 20 | W | 11/16 | look at cells, notice transformation efficiencies |
| 21 | M | 11/21 | immunostaining w/ Rabbit anti-K167 (Tom to bring – what concentration?)(red secondary) & Rat anti-alpha Tubulin (Green secondary) |
| 22 | M | 11/28 | nucleofect Guide 1 & Guide 2 (122), or just Guide 2 into WT HeLas. DON’T USE gfp-tubulin HeLas for this |
| 23 | W | 11/30 | Checked transfections in cover-slip bottom dish. Nucleofection effiiency Nuc blue |
| 24 | M | 12/5 | IF for Ki67 tubulin and dapi. seed glass bottom dish to view next meeting. treat cells with nocodazole at 10 am before class W |
| 25 | W | 12/7 | 1st hour: live cell imaging of nocodazole treated cells1 with NucBlue rest of class: work on posters |
| 26 | M | 12/12 | Poster presentations in ILC (RESERVE ROOM) |
- Make 10 mL of 3.3 nocodazole. 3 hours before class, remove 1 mL DMEM from each dish, replace with 1 mL 3.3uM nocodazole, for a final concentration of ~1.75 uM. Give students 1 mL aliquots of fluorobrite with 1.75 uM nocodazole and 20 drops/10 mL nucblue. ↩︎