10/13/10
- ✓ Transfection: HepG2 cells w/ Pc-ATF constructs
- ✓ HEK Gal4EED: induce 4-day wells with dox
Lipo transfection
> Note: HepG2 can be difficult to transfect. Try all constructs at first, optimize as needed.
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH126/MV2 |
2 μg |
4.1 μL |
4 μL |
500 μL
|
| 2 |
KAH154/MV2 |
" |
7.5 μL |
" |
"
|
| 3 |
KAH128/MV2 |
" |
4.0 μL |
" |
"
|
| 4 |
KAH157/MV2 |
" |
7.4 μL |
" |
"
|
| 5 |
KAH129/MV2 |
" |
4.1 μL |
" |
"
|
| 6 |
KAH156/MV2 |
" |
8.2 μL |
" |
"
|
| 7 |
KAH130/MV2 |
" |
5.7 μL |
" |
"
|
| 8 |
KAH158/MV2 |
" |
6.0 μL |
" |
"
|
| 9 |
KAH131/MV2 |
" |
3.9 μL |
" |
"
|
| 10 |
KAH159/MV2 |
" |
7.4 μL |
" |
"
|
| 11 |
KAH132/MV2 |
" |
4.4 μL |
" |
"
|
| 12 |
none |
--- |
--- |
" |
"
|
> 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 3.5 mL well (4 ml med. each); Grow cells at 37°C
> Results: All 11 constructs are expressed in about 1% of the cells. Cells are very clumpy, so this might prevent better transfection. Proceed with puromycin kill curve for HepG2 and stables.
| 
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