Serial Dilutions on Dv-1 Cells with Middle Band and P5 Concentrate DiNV Stocks for Virus Titering
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
| A |
|
cell control |
10^-1 |
10^-2 |
10^-3 |
10^-4 |
10^-5 |
|
| B |
|
500ul medium |
200ul 10-1 dilution |
200ul 10-2 dilution |
200ul 10-3 dilution |
200ul 10-4 dilution |
200ul 10-5 dilution |
|
| C |
|
500ul medium |
200ul 10-1 dilution |
200ul 10-2 dilution |
200ul 10-3 dilution |
200ul 10-4 dilution |
200ul 10-5 dilution |
|
| D |
|
500ul medium |
200ul 10-1 dilution |
200ul 10-2 dilution |
200ul 10-3 dilution |
200ul 10-4 dilution |
200ul 10-5 dilution |
|
| E |
|
500ul medium |
200ul 10-1 dilution |
200ul 10-2 dilution |
200ul 10-3 dilution |
200ul 10-4 dilution |
200ul 10-5 dilution |
|
| F |
|
|
|
|
|
|
|
|
- The same layout was used for each 48 well plate, one uses the middle band stock generated from the virus purification Kent and I tried, as well as the Passage 5 DiNV that went into the purification effort
- I was not sure what dilutions to do so I just went 10^-1 through 10^-5 and would do more if needed, there was no way to know how concentrated it was before this!
- Everything was done in the cell culture hood
- Prepared 50mL of serum free medium: 49.5mL schenider’s medium and 50ul gentamincin
- Prepared autoclaved 1.5mL tubes for dilitions
- Dilutions for both solutions were prepared in the same way, but were done separately so to not get confused
- Thawed aliquot of solution on ice
- Put 990ul serum free medium in 1.5mL tube
- Added 110ul virus stock to tube - 10^-1 tube
- Vortexed and spun down 10^-1 tube
- Added 990ul serum free medium to 4 more tubes and labeled for other dilutions
- Added 110ul of 10^-1 tube to the 10^-2 tube
- Vortexed and spun down the 10^-2 tube
- Added 110ul of 10^-2 tube to the 10^-3 tube
- Vortexed and spun down the 10^-3 tube
- Added 110ul of 10^-3 tube to the 10^-4 tube
- Vortexed and spun down the 10^-4 tube
- Added 110ul of 10^-4 tube to the 10^-5 tube
- Vortexed and spun down the 10^-5 tube
- Kept on ice before using
- Cleaned out a plastic tray with ethanol before putting in hood and filling with autoclaved paper towels
- Dumped out the fluid from 1 of the Dv-1 48 well plates into the tray and tapped down on PTs twice
- Added 200ul of either serum free medium or dilution (see layout above) to each well with cells
- Filled outer wells of the plates with DPBS
- This process was done for both plates, one with middle band DiNV solution and one with P5 DiNV solution
- Both plates were put in the 23C incubator for 48 hours