12/07/09
- ✓ Transfection: transfection and neo-induction of K-stop constructs
Transfection
> Test of Neo-inducible translation
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1-6 |
KAH5105/MV2 |
200 ng |
0.5 μL |
1.8 μL |
18.2 μL
|
| 7-12 |
KAH106/MV2 |
" |
0.4 μL |
" |
"
|
| 13-18 |
KAH107/MV2 |
" |
0.4 μL |
" |
"
|
| 19-24 |
KAH108/MV2 |
" |
0.4 μL |
" |
"
|
> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min.
> Add 6x vol DNA --> R.T./ 20 min.
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> After 4-5 hours, refresh medium and add varying amounts of neomycin (G418); make serial dilutions (1:2)
--> Initial dilution = 400 μg/ 100 μL (8 μL 50 mg/mL stock in 92 μL Opti-MEM)
--> 4x (= 400 μL) = 32 μL 50 mg/mL stock in 368 μL Opti-MEM
--> Final vol. of each dilution is 50 μL (4x = 200 μL)
| Wells |
Plasmid |
Neomycin (final conc.) |
Diluted Neo Vol.
|
| 1-6 |
KAH5105/MV2 |
0, 25, 50, 100, 200, 400 μg/mL |
50 μL
|
| 7-12 |
KAH106/MV2 |
" |
"
|
| 13-18 |
KAH107/MV2 |
" |
"
|
| 19-24 |
KAH108/MV2 |
" |
"
|
> Assay RFP after 18-24 hours
|
Project name
Main project page
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