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 min ; 95°C for 15 min ; Hold 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 )
