12/29/09
- ✓ Transfection 1: RFP-reporters KAH93-95 for time-lapse microscopy
- ✓ Transfection 2: RFP-reporters KAH93-95 for RNA(RT-PCR)
Transfection 1
> KAH93-95, 108/MV2 + JDS33-47 U2OS
> 12-well format, Fugene
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| Col.1 (1-3) |
KAH93/MV2 |
400 ng |
1.9 μL |
3.6 μL |
46.4 μL
|
| Col.2 (4-6) |
KAH94/MV2 |
" |
2.4 μL |
" |
"
|
| Col.3 (7-9) |
KAH95/MV2 |
" |
2.3 μL |
" |
"
|
| Col.4 (10-12) |
KAH108/MV2 |
" |
0.9 μL |
" |
"
|
> Master mixes (4, x3): 10.8 μL Fugene + 139.2 μL Opti-MEM --> R.T/ 5 min.
> Add 3x vol DNA --> R.T./ 20 min.
> Add 50 μL complexes to each well (2 ml med. each); Grow cells at 37°C overnight
Day 2
> Change to CO2 independent medium (L-15, no phenol red, 10% T.S. FBS, 1% P/S)
> Mark anchor point on well 1
> Set up plate under scope; secure plate to ensure no shifting; set to 34°C (Note: heater is +3-4°C off)
> Find RFP+ cells and set staging
> Add 1 μg/mL Dox; record images every 2-4 hours
Transfection 2
> KAH93-95/MV2 + JDS33-47 U2OS
> 6-well format, Lipofectamine 2000
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH93/MV2 |
2 μg |
9.4 μL |
4 μL |
500 μL
|
| 2 |
KAH93/MV2(Dox) |
" |
9.4 μL |
" |
"
|
| 3 |
KAH94/MV2 |
" |
12.0 μL |
" |
"
|
| 4 |
KAH95/MV2(Dox) |
" |
12.0 μL |
" |
"
|
| 5 |
KAH95/MV2 |
" |
11.3 μL |
" |
"
|
| 6 |
KAH95/MV2(Dox) |
" |
11.3 μL |
" |
"
|
> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Culture vol. = 3.5, no need to remove 500 mL. Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
Day 2
> Add 1μg/mL Dox to "Dox" wells.
Day 3
> RNA preps/ cDNA synthesis
| 
Project name
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