02/02/10
- ✓ Minipreps: KAH139-141/V0201
- ✓ Transfection: Gal4-EED expression constructs
- ✓ Thaw cells: HEK293 Luc #4 (to test Gal4-EED function)
Minipreps
> Check with XbaI digest
| Reagent |
Volume
|
Expected:Vector = 4500
1,2. KAH139/V0201 = 3655
1,2. KAH140/V0201 = 3599
1,2. KAH141/V0201 = 3187
|
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.
|
--> Combine duplicate samples, measure conc., and use for Lipo transfection
Lipo transfection
> Details
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH139/V0201 |
2 μg |
4.4 μL |
4 μL |
500 μL
|
| 2 |
" (+50 μg/mL neo) |
" |
4.4 μL |
" |
"
|
| 3 |
" (+100 μg/mL neo) |
" |
4.4 μL |
" |
"
|
| 4 |
" (+200 μg/mL neo) |
" |
4.4 μL |
" |
"
|
| 5 |
" (+400 μg/mL neo) |
" |
4.4 μL |
" |
"
|
| 6 |
" (+500 μg/mL neo) |
" |
4.4 μL |
" |
"
|
| 7 |
KAH140/V0201 |
" |
5.8 μL |
" |
"
|
| 8 |
KAH141/V0201 |
" |
5.1 μL |
" |
"
|
| 9 |
no DNA |
" |
--- |
" |
"
|
> 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.
> Starting vol. is 3.5 mL, no need to remove 500 μL; Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Add neomycin to samples 2-6
Day 2
> Do protein preps on all samples (for EED Western blot)
| 
Project name
Main project page
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