If you are doing knockout then direct transfection of sgRNA and cas9 protein is possible With lipofectamine 3000. The issue here is that there is no way to select the cells i.e. by puromycin so high transfection efficiency is required for the success.

Hi Anja,

I used Polyethyleneimine (PEI) to transfect my cells with the following protocol and got very satisfactory results.

For 6-well plate:

1. 2.5 μg/ml of plasmid DNA in 250 μl serum-free DMEM media [incubated for 10 minutes at room temperature]

2. 10 μl of PEI 1 µg/µl (linear MW 25,000) in 250 μl serum-free DMEM media [incubated for 10 minutes at room temperature]

3. Mix DNA and PEI, and incubate for 30 minutes at room temperature to form complex.

4. Replace each well’s media with 2 ml fresh DMEM media (without serum), and add the DNA/PEI mixtures to the wells dropwise all over the wells' surfaces.

5. Incubate 6-well plates at 37°C and 5% CO2 for 4 hours.

6. Replace each well’s media with 2 ml fresh complete medium.