Haynes Lab:Notebook/CRISPR Editing/2015/07/05

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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

Component Final vol / well
20uM siRNA duplex 2.5
Optimem 46
Oligofectamine 1.5
Total 50

After 4 hours, add 125ul DMEM + 3x serum to each well.