07/05/13
- HEK293 Gal4-EED transfection - for PcTF expression and flow cytometry
Lipo transfection
> Samples are for flow cytometry
> Seed new 6-well plates w/ ~5x105 cells/well in 3.4 mL plain medium (don't have any p.s.-free)
> Transfect human PcTF under control of constitutive CMV-TetO promoter (KAH160/MV1); 347 ng/μL; Note: this cell line has TetR, so constitutive CMV is needed.
Wells |
Plasmid |
DNA |
Volume + dH2O |
Opti-MEM |
PLUS |
Lipo |
|
1 |
KAH160/MV1 |
2 μg |
5.8 μL +14.2 |
570 μL |
2.5 μL |
7.5 μL
|
2 |
KAH160/MV1 |
2 μg |
5.8 μL +14.2 |
" |
" |
"
|
3 |
mock |
none |
0 μL + 20.0 |
" |
" |
"
|
4 |
none |
none |
--- |
--- |
--- |
---
|
> Add 570 μL Opti-MEM to each 20 μL DNA sample
> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp
> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp
> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)
> Grow cells at 37°C overnight
Results (7/06/13)
- Success! Very high levels of RFP expression in wells 1 & 2 (microscopy), zero signal in mock and untreated (3 & 4)
- Harvest cells for flow cytometry
|