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 Pc-TF Genomics
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06/12/13
- Gal4-EED/luc cells IFC - stain with anti-luc overnight
- PcTF transfection of U2OS
Gal4-EED/luc cells IFC
- Samples: first row of wells in the 24-well plate.
- Keep all others under PBS
- 0 μg/mL dox
- 0.1
- 0.2
- 0.5
- 1.0
- 2.0 (induced on 6/02/13, 10 days)
- Use 1:200 dilution of rabbit (poly) anti-luc (in 15 μL Gentex horse serum blocking solution per well)
- Incubate under parafilm "covers" overnight at 4°C in box with wet paper towels
Lipo transfection
> Cells were 100% confluent
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium
> Transfect human PcTF under control of constitutive CMV promoter (KAH160/MV1); 346.6 ng/μL
> Samples are for RT-PCR
| 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 |
" |
" |
"
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| 3 |
KAH160/MV1 |
2 μg |
5.8 μL +14.2 |
" |
" |
"
|
| 4 |
mock |
none |
0 μL + 20.0 |
" |
" |
"
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| 5 |
mock |
none |
0 μL + 20.0 |
" |
" |
"
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| 6 |
mock |
none |
0 μL + 20.0 |
" |
" |
"
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> 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)
> Add 1 μg/μL dox
> Grow cells at 37°C overnight
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Pc-TF Genomics
