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 PcTF Subcloning in E. coli
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06/06/2013
- ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
- ✓ Polycomb-ATF negative control lines: colony plates
Transfections
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:
- BD002 (3:1) Flp-in T-REx
- BD002 (4:1) Flp-in T-REx
BD002 (3:1) = 339 ng/μl
BD002 (4:1) = 317 ng/μl
PlpE = 192 ng/μl
| Plate-Well |
Plasmid |
DNA |
Volume |
dH2O |
Lipo |
PLUS reagent |
Opti-MEM (total)
|
| 1-1 |
BD002 (3:1) + FlpE |
1.5 + 0.5 μg |
4.42 + 2.60 μL |
12.98μL |
2.5μL |
7.5 μL |
570 μL
|
| 1-2 |
BD002 (3:1) + FlpE |
" |
4.42 + 2.60 μL |
12.98μL |
" |
" |
"
|
| 1-3 |
BD002 (4:1) + FlpE |
" |
4.73 + 2.60 μL |
12.67μL |
" |
" |
"
|
| 1-4 |
BD002 (4:1)+ FlpE |
" |
4.73 + 2.60 μL |
12.67μL |
" |
" |
"
|
| 1-5 |
BD002 (3:1) + --- |
" |
4.42 + --- |
15.58μL |
" |
" |
"
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| 1-6 |
BD002 (4:1) + --- |
" |
4.73 + --- |
15.58μL |
" |
" |
"
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- Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
- Label sterile microfuge (1.5 ml) tubes.
- Add 570 μL Opti-MEM to each 20 μL DNA sample.
- Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
- Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
- Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
- Incubate cells at 37°C in a CO2 incubator
- (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
Transgene expression should be detectable after 18 hours.
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)
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PcTF Subcloning in E. coli
