02/09/10
- ✓ U2OS Flp-in T-REx: add dox to Dox+ wells (6-well plate from 2/08)
- ✓ Transfection: Gal4-EED constructs in HEK293 luc reporter line
- ✓ Polycomb-ATF lines: frozen stocks for 90% confluent plates: 127-4, 128-8, 132-8; 1x 1:10 plate (per line)
Transfection
> HEK293 cells + Gal4-EED constructs (KAH139, 140, 141/V0201)
> Use Fugene, 24-well format
> Serial dilutions of plasmids: 800 - 25 ng in 5 μL
--> Start with 1600 ng in 10 μL OptiMEM
| Plasmid |
ng/μL |
Add (μL)... |
to OptiMEM (μL)
|
| 1. KAH139/V0201 |
454.2 |
3.5 |
7.5
|
| 2. KAH140/V0201 |
342.3 |
4.7 |
5.3
|
| 3. KAH141/V0201 |
388.8 |
4.1 |
5.9
|
--> Add 5 μL plasmid to 5 μL OptiMEM for each dilution step
| Wells |
Plasmid |
DNA |
Volume |
Fugene |
Opti-MEM
|
| 1-6 |
KAH139/V0201 |
25, 50, 100, 200, 400, 800 ng |
5.0 μL |
1.8 μL |
18.2 μL
|
| 7-12 |
KAH140/V0201 |
" |
" |
" |
"
|
| 13-18 |
KAH141/V0201 |
" |
" |
" |
"
|
| 19-24 |
no DNA |
" |
" |
" |
"
|
> Master mix: 90 μL Fugene + 910 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL Fugene mix to DNA --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay Luc after 18-24 hours
> Day 2
--> Refresh medium. Do luc assay next day (2/11).
|
Project name
Main project page
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