06/25/10
- ✓ Minipreps: KAH96, 142, 146, 147/V0201 DNAme-
- ✓ Transfection: KAH96, 142, 146, 147/V0201 DNAme-
- ✓ ChIP: cross-link FTRx and 154-2 cells
Minipreps
> DNA methylation-free plasmid preps for transfection (grown in C2925)
> Check KAH96, 142, 146, 147/V0201 with XbaI digest (add 641 to expected insert size)
| Reagent |
Volume
|
Expected:
V0201 band = 4516
Insert band = BB size + 641 bp
1-3. KAH96/V0201 = 3466
4,5. KAH142/V0201 = 5212
6-8. KAH146/V0201 = 7027
9-12. KAH147/V0201 = 8773
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| XbaI |
1.0
|
| dH2O |
10.5
|
| |
15 μL --> 37°C/ ~15 min.
|
Lipo transfection
> KAH96 [5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4]
> KAH142 [DPRE : 5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4]
> KAH146 [[KAH96] + EED expression cassette]
> KAH147 [[KAH142] + EED expression cassette]
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH96/ FlpE |
1.5, 0.5 μg |
1.5, 1.0 μL |
4 μL |
500 μL
|
| 2 |
KAH142/ FlpE |
1.5, 0.5 μg |
1.8, 1.0 μL |
" |
"
|
| 3 |
KAH146/ FlpE |
1.5, 0.5 μg |
2.0, 1.0 μL |
" |
"
|
| 4 |
KAH147/ FlpE |
1.5, 0.5 μg |
2.5, 1.0 μL |
" |
"
|
| 5 |
Lipo only |
--- |
--- |
" |
"
|
> 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.
> Add 500 μL complexes to each well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
|
Project name
Main project page
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