03/06/11
- ✓ Transfection: Pc-TF's into luc-silenced HEK cells (Fugene)
- ✓ KAH126-1: split back-up cultures; plate ~5x105 cells in a 6-well dish (3 dox-, 3 dox+) for RNA preps to optimize new RT-PCR primers
Fugene transfection
--> See 2/28/11
--> Use 6-day luc-silenced cells (plated last night)
- KAH160: human Pc-TF
- KAH170: deleted PCD
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1-6 |
KAH160/MV1 |
0, 50, 100, 200, 400, 800 ng |
2.5 μL = 800 ng |
1.8 μL |
18.2 μL
|
| 7-12 |
" |
" |
" |
" |
"
|
| 13-18 |
KAH170/MV1 |
0, 50, 100, 200, 400, 800 ng |
2.1 μL = 800 ng |
" |
"
|
| 19-24 |
" |
" |
" |
" |
"
|
> Add DNA to stdH2O for a total volume of 10 μL (2x master mix = 20 μL)
- KAH160: 10 DNA + 30 PBS --> Use 20 μL for serial dilution
- KAH170: 8.4 DNA + 31.6 PBS --> Use 20 μL for serial dilution
> Fugene master mixes (x2, 12 total): 3.6 μL Fugene + 36.4 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 30 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days and 4 days
> Note: (3/05/11) when plating cells, use total cells from two dox+ 10 cm plates (~40% confluent); also use ~10% of the cells (2 mL of 10 mL resuspension) to seed each new 10 cm plate (2 total) for continued dox treatment
|
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