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 Pc-TF Genomics
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6/30/13/dd/yy
- Gal4-EED/luc cells - split 6-well culture plate (shipment-2 cells) and treat 1 plate with dox
- PcTF transfection of SK-N-SH
- Gal4-EED ELISA set-up
Gal4-EED/luc cells IFC
- Previous trial, saw no difference in repression for 0.1, 0.2, 0.5, 1.0, 2.0 μg/mL dox. Try lower range.
- 1.0 μg/mL dox
- 10-1
- 10-2
- 10-3
- 10-4
- 0
Addition of serial dilutions of dox to cells
- Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary.
- Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes...
| |
tube 1 |
2 |
3 |
4 |
5 |
6
|
| 1 mg/mL dox (μL) |
8.0 |
--- |
--- |
--- |
--- |
---
|
-
|
mixture from prior tube (μL) |
--- |
100 |
100 |
100 |
100 |
---
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| +growth med. (μL) |
1000 |
900 |
900 |
900 |
900 |
---
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| Final [dox] (μg/mL) in 4 mL med. |
1.0 |
10-1 |
10-2 |
10-3 |
10-4 |
0
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Lipo transfection
> Samples are for RT-PCR
> 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-TetO promoter (KAH126/MV2); 226 ng/μL
; Note: no TetR in SK-N-SH, so expression will be constitutive
| Wells |
Plasmid |
DNA |
Volume + dH2O |
Opti-MEM |
PLUS |
Lipo |
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| 1 |
KAH126/MV2 |
2 μg |
8.8 μL +11.2 |
570 μL |
2.5 μL |
7.5 μL
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| 2 |
KAH126/MV2 |
2 μg |
8.8 μL +11.2 |
" |
" |
"
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| 3 |
KAH126/MV2 |
2 μg |
8.8 μL +11.2 |
" |
" |
"
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| 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)
> Grow cells at 37°C overnight
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Pc-TF Genomics
