07/31/11
- ✓ Transfection: for H3me reporter stable lines
Transfections: H3me reporters stables
> Lipofectamine LTX
> Follow Jason's protocol, 6-well plates
> Use ~1:3 ratio of FlpE plasmid to reporter plasmid
> Two different types of Flp-in T-REx: HEK293, U2OS
Plate 1: HEK293 FTRx
| Wells |
Reporter plasmid (CFP) |
1.5 μg DNA = |
Recombinase |
0.5 μg DNA =
|
| 1 |
208/V0200 |
10.4 μL |
FlpE |
1.0 μL
|
| 2 |
DPRE-208/V0200 |
6.2 μL |
FlpE |
1.0 μL
|
Plate 2: U2OS FTRx
| Wells |
Reporter plasmid (CFP) |
1.5 μg DNA = |
Recombinase |
0.5 μg DNA =
|
| 1 |
208/V0200 |
10.4 μL |
FlpE |
1.0 μL
|
| 2 |
DPRE-208/V0200 |
6.2 μL |
FlpE |
1.0 μL
|
Lipo LTX
> Dilute 2 μg DNA in dH2O (20 μL final)
> Add 580 μL Opti-MEM to 20 μL DNA
> Add 2.5 μL PLUS reagent --> R.T/ 5 min.
> Add 7.5 μL Lipo LTX --> R.T/ 30 min.
> Add ~600 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C
8/01/11
> Trypsinize cells w/ 0.5 mL medium; bring volume to 2 mL with selective medium
- U20S: McCoy's 5A complete (tet screened), 200μg/mL hygromycin, 15 μg/mL blasticidin
- HEK: DMEM complete (tet screened), 200μg/mL hygromycin, 15 μg/mL blasticidin
> Passage transfected and untransfected control cells to new plates...
- 100 μL cells, 10 mL, 10 cm dish
- 500 μL cells, 10 mL, 10 cm dish
- 1 mL cells, 4mL, 6-well plate (single well)
> Look for colony growth over time
|
Project name
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