Cas9 RNP nucleofection for cell lines using Lonza 4D Nucleofector
Bring 100 pmol of Cas9 to a final volume of 5 µL using Cas9 buffer ( 20 mM HEPES - KOH pH 7.5 , 150 mM KCl , 10 % glycerol , 1 mM TCEP ) .
For 40 µM stock : 2.5 µL .
Bring 120 pmol sgRNA to a final volume of 5 µL using Cas9 buffer .
This means you will need a minimum sgRNA concentration of 24uM .
Add Cas9 to sgRNA slowly while swirling pipette tip , should take 30s to 1 minute .
Allow RNP to form for 10 - 20 minutes .
Count cells .
( Trypsinize as needed . ) .
For each nucleofection , pipette 200k cells into a 15 mL conical .
Spin 100 x g for 10 minutes to pellet cells softly .
While the cells are spinning , prepare plate and cuvette .
Prepare a 12 - well - plate with 1mL media per well , and pre - warm in the incubator .
Prepare and label wells on 20uL nucleofection strips .
Configure Lonza 4d using recommended cell - type program .
Pipette off media from cells , gently but completely , using a P200 .
The pellet is very soft so be careful .
Resuspend cells in 20 µL of nucleofector solution ( usually SF media ) using a P200 .
Add the entire 10 µL RNP mix to the 20 µL resuspension and mix .
Add 1uL of 100uM donor DNA ( 100 pmoles ) and mix well .
Add nucleofection mixes to the multiwell cuvette , and cap .
Pay attention to the orientation of the cap and cuvette in the nucleofector , which is noted in the manufacturer’s instructions .
Insert cuvette into nucleofector and zap .
Allow cells to sit in nucleofection strips for 10 minutes post - nucleofection .
This is supposed to increase efficiency .
Add 80uL of pre - warmed media to each well .
Pipette mixture out with a P200 into your pre - warmed 12 - well plate .
This should get the vast majority of cells , but if you wish , you may wash out the rest with media from the same well , chemistry - style .
Allow cells 24 hours to settle and recover before attempted downstream analysis .
Consider including un - zapped controls to test viability .
