08/06/11
- Transfections: Test new activation domains on K. Hansen's HEK293 luc reporter system
Transfections: H3me test reporters (Gal4)
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates
> Use 1000 ng total plasmid (transcription factor plus empty vector)
> Plate 1 = repressed HEK 23;4;9 cells (5-day dox treatment, ~4 day wash-out)
> Plate 2 = non-repressed HEK 23;4;9 cells
Plate 1, 2
| Wells |
TF plasmid (RFP) |
Description |
200 ng DNA = |
Empty pcDNA3.1+ (MV1), 800 ng DNA =
|
| 1 |
KAH170/MV1 |
VP64-no DB |
0.5 μL |
0.8 μL
|
| 2 |
KAH60/pcVN |
VP64-Gal4DB |
0.4 μL |
"
|
| 3 |
KAH160/MV1 |
VP64-hPCD |
0.6 μL |
"
|
| 4 |
KAH220/MV1 |
SP1A-no DB |
0.5 μL |
"
|
| 5 |
KAH214/MV1 |
SP1A-Gal4DB |
0.6 μL |
"
|
| 6 |
KAH223/MV1 |
SP1A-hPCD |
0.5 μL |
"
|
| 7 |
KAH221/MV1 |
SP1B-no DB |
0.6 μL |
"
|
| 8 |
KAH215/MV1 |
SP1B-Gal4DB |
1.0 μL |
"
|
| 9 |
KAH224/MV1 |
SP1B-hPCD |
0.6 μL |
"
|
| 10 |
KAH221/MV1 |
p65-no DB |
1.3 μL |
"
|
| 11 |
KAH215/MV1 |
p65-Gal4DB |
0.7 μL |
"
|
| 12 |
KAH224/MV1 |
p65-hPCD |
0.9 μL |
"
|
Lipo LTX, Note: double each reaction
> Dilute 1 μg DNA in dH2O (10 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.
> Add 3 μL Lipo LTX --> R.T/ 30 min.
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C
> Express for two days; Luciferase assay early Monday morning
| 
Project name
Main project page
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