12/27/09
- ✓ Minipreps: KAH93, 94, 95/MV2 (from re-transformation colonies)
- ✓ Transfections: KAH120-125/MV2 (12-well format, Fugene)
Minipreps
> Check with E/P digests
| Reagent |
Volume
|
Expected:
1,2. KAH93/MV2 = 4839
3,4. KAH94/MV2 = 5011
5,6. KAH95/MV2 = 5083
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
3.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
8.5
|
| |
15 μL --> 37°C/ ~15 min.
|
Fugene transfection
> KAH120-125/MV2 + U2OS plain (12-well format, Fugene)
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1 |
KAH120/MV2 |
400 ng |
2.5 μL |
3.6 μL |
46.4 μL
|
| 2 |
KAH121/MV2 |
" |
5.3 μL |
" |
"
|
| 3 |
KAH122/MV2 |
" |
4.8 μL |
" |
"
|
| 4 |
KAH123/MV2 |
" |
4.0 μL |
" |
"
|
| 5 |
KAH124/MV2 |
" |
2.3 μL |
" |
"
|
| 6 |
KAH125/MV2 |
" |
5.0 μL |
" |
"
|
| 7 |
mock |
--- |
--- |
" |
"
|
> 3.6 μL Fugene + 46.4 μL Opti-MEM --> R.T/ 5 min.
> Add DNA --> R.T./ 20 min.
> Add 50 μL complexes to each well (2 ml med. each); Grow cells at 37°C overnight
Day 2
> Check RFP/CFP expression
--> Experiment looks interesting so far. Document images and repeat again with full set of 9 pairs when KAH117-119 are finished
|
Project name
Main project page
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