12/21/09
- ✓ Transfections: Polycomb ATF's for stable lines
Transfections
> U2OS Flp-in T-REx cells + Polycomb-ATF plasmids (puro)
> Use Lipofectamine, 6-well format
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH126/MV2 |
2 μg |
4.1 μL |
4 μL |
500 μL
|
| 2 |
KAH127/MV2 |
" |
4.0 μL |
" |
"
|
| 3 |
KAH128/MV2 |
" |
4.0 μL |
" |
"
|
| 4 |
KAH129/MV2 |
" |
4.1 μL |
" |
"
|
| 5 |
KAH130/MV2 |
" |
5.8 μL |
" |
"
|
| 6 |
KAH131/MV2 |
" |
3.9 μL |
" |
"
|
| 7 |
KAH132/MV1 |
" |
4.4 μL |
" |
"
|
| 8 |
KAH133/MV1 |
" |
3.9 μL |
" |
"
|
| 9 |
no DNA |
--- |
--- |
" |
"
|
| 10 |
untreated |
--- |
--- |
--- |
"
|
> 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.
> Remove 500 μL medium from each well; Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Grow for two days, then plate dilution cultures in selection medium (puro)
| 
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