12/06/09
- ✓ Transfection: KAH93, 94, 95/MV2 (RFP); KAH99, 100, 101/MV2 (CFP)
Transfections
> U2OS cells + Gal4DB-PEST-RFP constructs
> Use Lipofectamine, 6-well format
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH93/MV2 |
2 μg |
4.4 μL |
4 μL |
500 μL
|
| 2 |
KAH94/MV2 |
" |
4.8 μL |
" |
"
|
| 3 |
KAH95/MV2 |
" |
8.7 μL |
" |
"
|
| 4 |
KAH99/MV2 |
" |
15.2 μL |
" |
"
|
| 5 |
KAH100/MV2 |
" |
10.4 μL |
" |
"
|
| 6 |
KAH101/MV2 |
" |
7.5 μL |
" |
"
|
| 7 |
KAH36(control) |
" |
2.5 μL |
" |
"
|
| 8 |
mock |
--- |
--- |
" |
"
|
| 9 |
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.
~~Cells in 6-well plates too crowded, discard; do same-day plating/ transfection from back-up stocks~~
> Harvest cells, plate 5x105 cells/3.5 ml ab-free medium in 6-well plates
> Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Check RFP & CFP expression (microscopy) after 18-24 hours
|
Project name
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