08/30/10
- ✓ Transfection: Pc-ATF's into H3me3 reporter cells
- ✓ Freeze-down: KAH142-3
- ✓ ChIP/ Co-IP: x-link KAH130-4, 131-4 (formaldehyde x-link only, no DMA)
Fugene transfection
> Transfect the following cell lines (in 12-well plates, 6 wells per line):
- KAH96-11
- KAH142-3
- KAH142-5
- KAH146-1
with six different Pc-ATF constructs and neg. ctrls. (dox inducible). Test to see if Pc-ATF can activate YFP.
> Pc-ATF test constructs (human only for this trial):
- KAH126: human PCD-ATF
- KAH154: human PCD-RFP (no act. domain)
- KAH130: human CBX8-ATF
- KAH158: human CBX8-RFP (no act. domain)
- KAH132: ATF (no Polycomb)
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1 |
KAH126/MV2 |
200 ng |
0.4 μL |
1.8 μL |
18.2 μL
|
| 2 |
KAH154/MV2 |
" |
2.0 μL |
" |
"
|
| 3 |
KAH130/MV2 |
" |
0.4 μL |
" |
"
|
| 4 |
KAH158/MV2 |
" |
0.6 μL |
" |
"
|
| 5 |
KAH132/MV2 |
" |
0.4 μL |
" |
"
|
| 6 |
none |
0 |
--- |
" |
"
|
Use the same set-up (above) for:
1-6: KAH96-11 cells
7-12: KAH142-3 cells
13-18: KAH142-5 cells
19-24: KAH146-1 cells
> Master mixes (4, 4x mixes): 7.2 μL Fugene + 72.8 μL Opti-MEM --> R.T/ 5 min.
> Add 4x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (2 ml med. each); Grow cells at 37°C overnight
> Tomorrow, photograph RFP/YFP without dox, then add dox and check for RFP/YFP in the evening
| 
Project name
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