02/17/10
- ✓ Transfection: for RFP-repressor knock-down experiment
Lipo transfection
> Wells 1-9: JDS33-47 cells; Wells 10-12: U2OS (Flp-in T-REx) cells
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH94/MV2 |
2 μg |
6.2 μL |
4 μL |
500 μL
|
| 2 |
KAH94/MV2(24hr dox) |
" |
6.2 μL |
" |
"
|
| 3 |
KAH94/MV2(48hr dox) |
" |
6.2 μL |
" |
"
|
| 4 |
5xluc-/MV2 |
" |
4.0 μL |
" |
"
|
| 5 |
5xluc-/MV2(24hr dox) |
" |
4.0 μL |
" |
"
|
| 6 |
5xluc-/MV2(48hr dox) |
" |
4.0 μL |
" |
"
|
| 7 |
no DNA |
--- |
--- |
" |
"
|
| 8 |
no DNA(24hr dox) |
--- |
--- |
" |
"
|
| 9 |
no DNA(48hr dox) |
--- |
--- |
" |
"
|
| 10 |
KAH94/MV2 |
2 μg |
6.2 μL |
" |
"
|
| 11 |
5xluc-/MV2 |
" |
4.0 μL |
" |
"
|
| 12 |
no DNA |
--- |
--- |
" |
"
|
> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Add 500 μL complexes to each well (4 ml final vol.); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Wells 3, 6, 9 (48hr dox): Add dox, final = 1 μg/mL
Day 2
> Wells 2, 5, 8 (24hr dox): Add dox, final = 1 μg/mL
Day 3
> Flow cytometry analysis of RFP and YFP expression
| 
Project name
