10/27/10
- ✓ Transfections: HEK293 Gal4EED + Pc-ATF's
Transfections
> Try transfecting with Pc-ATF first, then inducing H3K27me3
> HEK293 Gal4-EED 23;4;9 cells, no dox induction
> Use Fugene, 12-well format, 1.5 mL medium per well
> 2 plates total (1 control, 1 for dox induction time course)
Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
1. |
KAH126/MV2 |
500 ng |
1.0 μL |
1.5 μL |
48.5 μL
|
2. |
KAH154/MV2 |
" |
2.0 μL |
" |
"
|
3. |
KAH128/MV2 |
" |
1.0 μL |
" |
"
|
4. |
KAH157/MV2 |
" |
2.0 μL |
" |
"
|
5. |
KAH129/MV2 |
" |
1.0 μL |
" |
"
|
6. |
KAH156/MV2 |
" |
2.0 μL |
" |
"
|
7. |
KAH130/MV2 |
" |
1.5 μL |
" |
"
|
8. |
KAH158/MV2 |
" |
1.5 μL |
" |
"
|
9. |
KAH132/MV2 |
" |
1.0 μL |
" |
"
|
10. |
KAH60/pcVN |
" |
1.0 μL |
" |
"
|
11. |
no DNA |
--- |
--- |
" |
"
|
> Master mix (2x, 1 per plate): 3 μL Fugene + 97 μL Opti-MEM --> R.T./ 5 min.
> Add 2x vol. DNA --> R.T./ 20 min.
> Remove 0.5 mL medium from each well (decrease volume to improve transfection efficiency)
> Add 50 μL DNA/Fugene mix to each well (1.5 ml medium/ well); Grow cells at 37°C
> Check RFP after 18-24 hours
|