Transfected siRNAs EZH2 and SUZ12 into gal4-EEDs with and without dox
Cells plated yesterday looked good.
- Cells plated at 30% were at about 80%
- Cells plated at 10% and 20% were at about 50%
Transfected 2 wells for each siRNA and each cell type
Resuspended duplex siRNAs with 100ul of unopened, RNase-free water for final concentration 20uM. Used RNase-away to reduce risk of degradation.
Mix optimem and siRNA
- 11ul siRNA + 176ul Optimem in 1.5mL tube
Mix oligofectamine and optimem
- 14.25ul oligofectamine + 57ul optimem
- let stand for 10 minutes
Mix DNA and oligofectamine
- add 33ul oligofectamine to tubes with DNA
- let stand for 30 minutes
While complexes form, prep cells
- Removed media
- Wash in 100ul PBS
- Aspirate PBS
- Add 200ul of plain DMEM
Add complexes to cells
- Add 50ul complexes dropwise to each well
||Final vol / well
| 20uM siRNA duplex
After 4 hours, add 125ul DMEM + 3x serum to each well.