07/24/10
- ✓ Transfections: Test Gal4-ATF's on KAH142 H3me reporter clones
Fugene transfection
> 12-well plate
1-4. KAH142-1
5-8. KAH142-3
9-12. KAH142-5
Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
1,5,9 |
no plasmid |
--- |
0 μL |
1.8 μL |
18.2 μL
|
2,6,10 |
KAH54/pcVN |
" |
0.4 μL |
" |
"
|
3,7,11 |
KAH59/pcVN |
" |
0.6 μL |
" |
"
|
4,8,12 |
KAH60/pcVN |
" |
0.4 μL |
" |
"
|
> Master mixes (3x mix, 4 mixes): 5.4 μL Fugene + 54.6 μL Opti-MEM --> R.T/ 5 min.
> Add 3x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37°C overnight
7/25/10
Results:
- Background YFP expression: two or three cells for KAH142-5, none for KAH142-3, KAH142-1 control well was contaminated
- KAH54/pcVN (Gal4DB-VP64): YFP+ cells in KAH142-3
- KAH59/pcVN (Gal4DB-mCh-mCh-VP64): RFP+/YFP+ cells in KAH142-3 and KAH142-5
- KAH60/pcVN (Gal4DB-mCh-VP64): strange result; RFP+/YFP- or RFP-/YFP+ cells
Conclusions:
- Keep KAH142-3 and 142-5 for frozen stocks; use 142-3 for further experimentation
- Use KAH59/pcVN to screen other H3me Reporter clones
|