| 9/19 |
triple stained LLCPk slides: DAPI, rat anti-alpha tubulin & anti-rat-FITC, red phalloidin |
| 9/26 |
thaw LL flask |
| 9/27 |
change medium |
| 9/28 |
split into large flask |
| 9/30 |
put 5 x 10^5 cells into each of 9 flasks |
| 10/1 |
students nucleofect |
| 10/3 |
Start G418 selection of transfected LLs |
| 10/9 |
Thaw 3t3 into DMEM, spin down, resuspend, plate on coverslips. No adherence (or all dead?) Make new DMEM, replace medium in flasks |
| 10/10 |
Stop selection - cells look bad. Replace selection medium with regular F10Hams |
| 10/11 |
very few adherent cells. |
| 10/15 |
Thaw LL-GFP-alphas,HeLa-H2B-GFP, Mcherry alpha tubulin |
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(get HeLa GFP alpha tubulin from Pat) |
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3t3s look fine |
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split transfected cells into one 25 cm2 flask to grow up, one 12.5 cm2 flask to continue selection |
| 10/16 |
Plate in coverslip dishes for lab on 10/17 (had to be done in Pat's lab) |
| 10/17 |
live cells for mitosis (made in Pat's lab: HeLa-GFP-tubulin, LLCPk-GFP tubulin) |
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thaw LLCPk-GFP-actin for use in motility lab 10/22 |
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thaw LLCPk-GFP-tubulin to test freeze/thaw protocol |
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split 3t3s to grow up for live cell observations 10/22 |
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split LL parentals in case anyone wants to repeat nucleofection |
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split nucleofected cells for students; they take over care of them 10/24 |
| 10/21 |
plate (coverslip bottom dishes) 3t3, LL-GFP-actin from flasks |
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make big flask of LL-GFP-actin to freeze |
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thaw b16, spin down, resuspend in DMEM, plate (coverslip dishes) |
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froze 3 vials HeLa-GFP-H2b/mcherry-tubulin |
| 10/22 |
Bioimaging students split their 2 flasks of nucleofected cells (G418/unselected); made 2 new flasks, 2 coverslip dishes. |
| 10/23 |
Set up sterile hoods with all equipment students will need. |
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Freeze 3 vials of 3t3s |
| 10/24 |
students split nucleofected cells |
| 10/25 |
split LL-GFP-tub, LL-GFP-actin and freeze 2 vials each |
|
split HeLa-GFP-tub: very strange - some cells never balled up. |
| 10/28 |
split LL-GFP-tub: 1 flask with 500,000 for nucleofection 10/29, one to keep culture going |
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split better looking parentals just to keep going |
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HeLas look OK, but sparse |
| 10/29 |
students split cells, work on projects |
| 10/30 |
make 1 flask of LL parentals to nucleofect, 1 flask of LL-GFP-tubulin (plus 1 flask each to perpetuate) |
| 10/31 |
students work on their cells |
| 11/01 |
inner incubator door was open, temp was 28C, CO2 was 3 |
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replace medium on HeLas (cultures are pretty sparse) |
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replace medium on parentals |
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split LL-GFP-actin to 12.5 flask |
| 11/04 |
plate 2 coverslip dishes of parental LL's for ER Tracker treatment |
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make 2 12.5 flasks for propagation |
| 11/05 |
|
| 11/06 |
split LL-GFP-tubulin: 2 full flasks for nucleofection, one for propagation |
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aliquot 40 mL trypsin |
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freeze 3 vials HeLa-GFP-tubulin |
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one flask HeLa-GFP-tubulin in case students want it |
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split flask of LL-GFP-actin in case students want it |
| 11/08 |
Kate sick |
| 11/10 |
split parentals |
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split LL-GFP-actin |
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refeed HeLa-GFP-tubulin |
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aliquot new F10-Hams & DMEM |
| 11/11 |
Split BL & AM nucleofected cells: 1 flask for nucleofection, one for propagation |
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G418 selection @ 2 mg/mL |
| 11/12 |
students work on cultures |
| 11/13 |
plate LL-GFP tubulin for nucleofection on 11/14 (with students) |
| 11/14 |
pat brings over IF LL's (intermediate filament eGFP vimentin) |
| 11/15 |
check IF flasks: all light. Split on Monday |
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split LLCPK parentals |
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split HeLa-GFP-tubulin |
| 11/18 |
8:30 am help students (AZ) count to prep cells (LL-IF-GFP) for nucleofection |
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split IF flasks for freezing (2 75cm2 flasks), student projects (2 25 cm2 flasks- each with 500,000 cells in case anyone wants to nucleofect them on Tuesday) |
| 11/19 |
split LL-GFP-tubulin: 1 heavy, 1 light |
| 11/20 |
split heavy LL-GFP-tubulin, count, put 500,000 in flask for Sam & Brian to nucleofect |
| 11/21 |
students work |
| 11/22 |
LLCPK GFP-IF: freeze 3 (2?) vials from T75 flask; split into T25 flask "in case" |
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LLCPK GFP-tubulin: split light |
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J & G: split; will split again 11/29 to make a nucleofection flask 12/1 |
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LLCPK Lifact Actin 488 split into T75 flask for freezing; small flask "in case" |
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KTRM: change medium in dish and flask |
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LLCPK parentals: split lightly "in case" |
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HeLa GFP-Tubulin split lightly "in case" |
| 11/25 |
ASBK split 2 flasks |
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KTRM: |
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GOJC H2B: split |
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Z&A: actin split 1 flask + G418, |
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Z&A IF change medium plut G418 |
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SLBM split GFP-Tub 2 |
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AMBL IF+Actin split plus G418 reduced by half |
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SACW will come in to do their own |
| 11/27 |
Freeze LifAct-488 LL |
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SLBM split "s1" (or wait till 11/29?) |
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SLBM split s2 flask LL-red-mitochondria to a 75 flask to freeze Friday |
| 11/29 |
LLGFP-GFP-tub split (so students can split on 12/1 for nucleofection 12/2) |
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SLBM freeze T75 (get labels from Sam & Brian) |
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HeLas look OK, but sparse |