Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporation
Seed the cells so that they will be around 70-90% confluent on the day of transfection.
Set up the RNP formation reaction as follows below:
Component7 µl reactionResuspension Buffer R5.8 µlEnGen Cas9-NLS (20 µM)0.6 µlsgRNA (20 µM)0.6 µl. 
Gently mix the reaction and incubate at room temperature for 20 minutes.
During the incubation, trypsinize the cells, washing once to remove any traces of trypsin.
Remove 1-2 x 106 cells to a sterile microfuge tube.
(One tube of cells should be enough for 10 transfections).
Resuspend the cells in 10 ml of media and count.
Pellet for 5 min at 500 x g.   
Wash the cells once with 1X PBS.
Resuspend the cells in 50 µl of Resuspension Buffer R. 
Prepare a 24-well plate by adding 500 µl growth medium to the appropriate number of wells.
Add 5 µl of cells to each 7 µl RNP reaction.
Aspirate 10 µl of the RNP/cells mix into a 10 µl Neon tip.
Electroporate the cells under the following conditions: 1700V, 20 ms, 1 pulse.
Immediately transfer the cells to the prepared 24-well plate.
Incubate the cells in a humidified 37 °C, 5% C02 incubator for 48-72 hours.
Gently aspirate the media from the cells.
Add 75 µl of Epicentre QuickExtract™ DNA Extraction Solution and shake/vortex for 5 minutes.
Transfer the solution to a PCR plate or tubes and place in a thermocycler, running the following program:
65°C for 15 min95°C for 15 minHold at 4°C. 
Dilute the DNA 1:10 in nuclease-free water. 
Follow the protocol detailed in the EnGen Mutation Detection Kit (E3321S) manual Pellet for 5 min at 500 x g.   
Wash with 100 µl 1X PBS. (1/2) 
Wash with 100 µl 1X PBS. (2/2)
