Electroporation of Cas9 protein into human pluripotent stem cells

Add 10µL to a sterile 1.5 ml tube. Add 6µg. Then add 1.2µg. Pipet up and down to mix. Let it sit at Room temperature for 0h 10m 0s. This is enough for 2 transfections (== one 6 well).

While waiting for the Cas9 to bind to sgRNA, individualize cells with Accutase. Neutralize Accutase with 5x volume E8 with Rock inhibitor.

Count cells. You will need 2x10⁵ for each transfection.

Spin down cells. Let it sit for a while so all the residue media can go down to the bottom of the tube. If the residue media is too much, take it out with a P200 pipet.

Resuspend cells to a concentration of 2x10⁵ per 5 μl (ie 4x10⁷ per ml) using buffer R.

Prepare a 24 well matrigel coated plate. Add 0.5mL-1mL to the wells you will use. Add HAS (1:2500) to each well. Each transfection goes into one well.

Wipe the Neon pipet station with EtOH and place it inside the hood.

Add 3mL to the neon tube. Place the tube inside the station. You should feel a click before the tube is securely seated in the station.

Use program 13 from the optimization tab for electroporation parameter. Program 9 should also work.

When everything is ready, mix 10µL-11µL with the Cas9+RNA containing R buffer. The final volume should be in the range of 21µL-22µL.

Take up a neon tip, pipet 10µL and electroporate with program 13.

If you see air bubble in the tip, take it out, push everything out of the tip and repipet the mixture.

If you see sparking during the electroporation, your efficiency will reduce significantly.

Once electroporation is complete, push everything into one well of a 24 well plate. Do not pipet up and down with Neon tip.

Repeat the same procedure with the same tip and the left over cell mixture. This is just a replicate.

Disperse cells evenly in the well and place cells in a low O2 incubator.

Put electroporated cells into low oxygen incubator for 2 days (Rm 329, key can be found at Melissa’s desk).