12/18/09
- Transfection: transfection and neo-induction of K-stop constructs
Transfection
> Test of Neo-inducible translation in U2OS (plain) cells
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1-6 |
KAH105/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 overnight
Day 2
> Check RFP expression
--> Much less RFP in K-stop samples than K controls; some leaky K-stop expression (recorded images)
> Refresh medium and add varying amounts of neomycin (G418)
--> Make neo dilutions in 50 μL Opti-MEM (see table)
| Wells |
Transfection |
Neomycin (final conc. in well) |
50 mg/mL neo Vol. |
Opti-MEM
|
| 1-6 |
KAH5105/MV2 |
0, 50, 100, 200, 400, 500 μg/mL |
0, 1, 2, 4, 8, 10 μL |
50 μL
|
| 7-12 |
KAH106/MV2 |
" |
" |
"
|
| 13-18 |
KAH107/MV2 |
" |
" |
"
|
| 19-24 |
KAH108/MV2 |
" |
" |
"
|
> Assay RFP after 18-24 hours
--> Checked after 48 hours on 12/21/09(snowstorm on Sunday); indication of induction in some cells
--> Move forward with neo-inducible Gal4-EED; quantify by Western blot
|
Project name
Main project page
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