08/08/2013
Gal4-VP64, Cyan Transactivation
1 day before transfection:
- Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.
- 22.5mL U2OS media (p/s) + 2.5mL trypsinized cells from T-75 flask with 100% confluent cells. Incubate 6-well plate overnight.
Transfections
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: BD002 co-transfect with Gal4-VP64 (10:1); hygro resistance replaces zeo resistance after transfection
> Use Lipofectamine, 6-well format
> Plasmids:
- BD002 = 346.61 ng/μl
- Gal4-VP64 transactivation plasmids
- KAH59 (MV5) = 277.719
- KAH54 (PCVN) = 217.267
- KAH59 (PCVN) = 203.300
- KAH60 (PCVN) = 169.931
- KAH66 (MV5) = 174.819
Plate-Well |
Plasmid |
DNA |
Volume |
dH2O |
Lipo |
PLUS reagent |
Opti-MEM (total)
|
1-1 |
BD002 + KAH59(PcVN) |
1.81 + 0.19 μg |
5.23 + 0.89 μL |
13.88μL |
2.5μL |
7.5 μL |
570 μL
|
1-2 |
BD002 + KAH60(PcVN) |
" |
5.23 + 1.07 μL |
13.7μL |
" |
" |
"
|
1-3 |
BD002 + KAH54(PcVN) |
" |
5.23 + 0.83 μL |
13.94μL |
" |
" |
"
|
1-4 (-)Ctrl |
BD002 + KAH59(MV5) |
" |
5.23 + 0.65 μL |
14.12μL |
" |
" |
"
|
1-5 |
BD002 + KAH66(MV5) |
" |
5.23+1.04 |
13.73μL |
" |
" |
"
|
- Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
- Label sterile microfuge (1.5 ml) tubes.
- Add 570 μL Opti-MEM to each 20 μL DNA sample.
- Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
- Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
- Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
- Incubate cells at 37°C in a CO2 incubator
- To reduce toxicity, after 6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
Transgene expression should be detectable after 18 hours.
|