Taking Samples and Fluid Changing Flasks at the 12 Week Mark for Experimental DiNV Evolution in Dinn Cells (~84 days)
Sampling 20231219
Cells were inoculated on the 26th of Sept see post here. There are 3 replicate flasks that have been left to grow and get infected in the 23C incubator for 84 days. I am only removing supernatant for samples, the cells are not getting passed throughout this experiment, the only thing that happens is a tri-weekly fluid change (once every three weeks). In the six week sampling I took 7 50ul supernatant and 1 1mL supernatant sample per replicate flask.
All steps took place in the cell culture hood
- Made fresh 10% FBS, 4% mushroom Schneider’s medium with antibiotics in the hood to room temperature
- Prepared 1.5mL tubes to take samples
- Removed 7 samples of 50ul and 1 sample of 1000ul from each flask before changing fluid and placed into the 1.5mL tubes:
| tube number | day sampled | date sampled | sample volume | replicate tube | flask from |
|---|---|---|---|---|---|
| 66 | day 84 | 20231219 | 50ul | 1 | A |
| 67 | day 84 | 20231219 | 50ul | 2 | A |
| 68 | day 84 | 20231219 | 50ul | 3 | A |
| 69 | day 84 | 20231219 | 50ul | 4 | A |
| 70 | day 84 | 20231219 | 50ul | 5 | A |
| 71 | day 84 | 20231219 | 50ul | 6 | A |
| 72 | day 84 | 20231219 | 50ul | 7 | A |
| 73 | day 84 | 20231219 | 1mL | NA | A |
| 74 | day 84 | 20231219 | 50ul | 1 | B |
| 75 | day 84 | 20231219 | 50ul | 2 | B |
| 76 | day 84 | 20231219 | 50ul | 3 | B |
| 77 | day 84 | 20231219 | 50ul | 4 | B |
| 78 | day 84 | 20231219 | 50ul | 5 | B |
| 79 | day 84 | 20231219 | 50ul | 6 | B |
| 80 | day 84 | 20231219 | 50ul | 7 | B |
| 81 | day 84 | 20231219 | 1mL | NA | B |
| 82 | day 84 | 20231219 | 50ul | 1 | C |
| 83 | day 84 | 20231219 | 50ul | 2 | C |
| 84 | day 84 | 20231219 | 50ul | 3 | C |
| 85 | day 84 | 20231219 | 50ul | 4 | C |
| 86 | day 84 | 20231219 | 50ul | 5 | C |
| 87 | day 84 | 20231219 | 50ul | 6 | C |
| 88 | day 84 | 20231219 | 50ul | 7 | C |
| 89 | day 84 | 20231219 | 1mL | NA | C |
- Removed 1.5mL of remaining medium in each flask and discarded it into a 15mL conical. The conical was placed in the autoclave trash when full (should contain virus)
- Added 3mL of 10% FBS, 4% mushroom Schneider’s medium with antibiotics to each flask gently
- Gently rocked flasks to distribute the medium
- Placed flasks back in the 23C incubator for another 3 weeks
- Sample tubes were frozen at -20
Sample information can be found here
Note that I noticed the flasks looked pretty bad at this point, lots of floating cells and almost no cells between the clumps of cells. This experiment might need to end soon.
Because of the holidays, I did not extract DNA from samples immediately after sampling.