04/09/11
- ✓ Transfection: Pc-TF's + luc-repressed HEK's (2 day w/o)
Fugene transfection
- KAH160: human Pc-TF
- KAH165: human PCD
- KAH161: fly Pc-TF
- KAH167: fly PCD
- KAH162: fish Pc-TF
- KAH166: fish PCD
- KAH170: deleted PCD
- mock (Fugene)
--> Plate 1: no dox
--> Plate 2: Dox-treated cells (2 days, wash-out day 2)
--> Plate 3: Dox-treated cells (4 days, wash-out day 2)
--> Plate cells: Collect cells from 2 10cm plates, resuspend in 25 mL medium, aliquot 1 mL per well in 24-well plate
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1-3 |
KAH160/MV1 |
100, 200, 400 ng |
1.2 μL = 400 ng |
1.8 μL |
18.2 μL
|
| 4-6 |
KAH165/MV1 |
" |
0.8 μL = 400 ng |
" |
"
|
| 7-9 |
KAH161/MV1 |
" |
1.0 μL = 400 ng |
" |
"
|
| 10-12 |
KAH167/MV1 |
" |
1.0 μL = 400 ng |
" |
"
|
| 13-15 |
KAH162/MV1 |
" |
1.1 μL = 400 ng |
" |
"
|
| 16-18 |
KAH166/MV1 |
" |
0.9 μL = 400 ng |
" |
"
|
| 19-21 |
KAH170/MV1 |
" |
1.0 μL = 400 ng |
" |
"
|
| 22-24 |
mock |
" |
0 |
" |
"
|
> Add DNA to stdH2O for a total volume of 5 μL (3x master mix = 15 μL)
--> Serial dilutions: start w/ 6x DNA + dH2O = 30 μL --> Use 15 μL for serial dilutions
> Fugene master mixes (x3, 24 total): 5.4 μL Fugene + 54.6 μL Opti-MEM --> R.T/ 5 min.
> Add 15 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days
|
Project name
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