04/23/10
- ✓ Transfections: H3me reporters into cell lines KAH126-1, 130-2, 132-8
- ✓ Polycomb-ATF negative control lines: colony plates
Transfections
> U2OS Flp-in T-REx lines (puro/zeo/blast) + Polycomb-ATF plasmids (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:
- KAH126-1 Flp-in T-REx
- KAH130-2 Flp-in T-REx
- KAH132-8 Flp-in T-REx
| Plate-Well |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1-1 |
KAH96/V0201 + FlpE |
1.5 + 0.5 μg |
2.4 + 1.4 μL |
4 μL |
500 μL
|
| 1-2 |
KAH142/V0201 + FlpE |
" |
2.4 + 1.4 μL |
" |
"
|
| 1-3 |
KAH146/V0201 + FlpE |
" |
6.4 + 1.4 μL |
" |
"
|
| 1-4 |
KAH147/V0201 + FlpE |
" |
9.6 + 1.4 μL |
" |
"
|
| ---
|
| 2-1 |
KAH96/V0201 + FlpE |
1.5 + 0.5 μg |
2.4 + 1.4 μL |
4 μL |
500 μL
|
| 2-2 |
KAH142/V0201 + FlpE |
" |
2.4 + 1.4 μL |
" |
"
|
| 2-3 |
KAH146/V0201 + FlpE |
" |
6.4 + 1.4 μL |
" |
"
|
| 2-4 |
KAH147/V0201 + FlpE |
" |
9.6 + 1.4 μL |
" |
"
|
| ---
|
| 3-1 |
KAH96/V0201 + FlpE |
1.5 + 0.5 μg |
2.4 + 1.4 μL |
4 μL |
500 μL
|
| 3-2 |
KAH142/V0201 + FlpE |
" |
2.4 + 1.4 μL |
" |
"
|
| 3-3 |
KAH146/V0201 + FlpE |
" |
6.4 + 1.4 μL |
" |
"
|
| 3-4 |
KAH147/V0201 + FlpE |
" |
9.6 + 1.4 μL |
" |
"
|
> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min.
> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Grow for one/two days, then plate dilution cultures in selection medium (+hygro)
Polycomb-ATF negative control lines: colony plates
> For each line make 2 plates: ~1x105 and 5x104 cells
> 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo
> Also make 6-well back-up plate for all transfections (~3x105 cells in selection medium)
| 
Project name
Main project page
Previous entry Next entry
