RNP formation and nucleofection
-Duplex and RNP formation
Resuspend crRNA and tracrRNA in IDTE buffer or IDT duplex buffer to generate 200 µM stocks (keep on ice, store at -20°C)
crRNA: resuspend 2nmol in 10ul IDT duplex buffer tracrRNA:resuspend 5nmol in 25ul IDT duplex buffer
Prepare gRNA duplexes (10 µl 80 µM):
4 µl crRNA
4 µl tracrRNA-Atto-550
2 µl IDT duplex buffer
5 min @ 95 °C, let cool down to RT (thereafter keep on ice and store at -20 °C)
-Prepare RNPs
Per nucleofection add 1.55 µl sgRNA duplex to 1.05 µl IDT HiFi Cas9
Incubate for 20 min at RT
-Nucleofection
Per nucleofection reaction combine 82 µl nucleofection buffer in the fridge (depending on cell line) with 18 µl nucleofection buffer supplement
Harvest and count cells, (with hemocytometer: average of 4 corner squares* dilution factor*10,000/ml), 200k-300k per reaction, wash once with PBS
Prepare transfection mixes (add 2.6 µl RNP and/or 2 µg donor plasmid to 50 µl nucloefection buffer plus supplement)
4 ul of alfa tagged/ flag tagged plus one 1ul of 1ug/ul GFP as a control
Add cell suspension to transfection mix, and transfer to nucleovette, no bubbles while transferring
Run nucleofection program on Lonza 4D nucleofector (for N2102EP), select nucleovette, select the program and pulse code (CA, 137).
Add 500 µl of culture medium to nucleovette and transfer cells to culture dish using plastic pippets to the 12 well plates containing 500 ul media.
Incubate at 37 °C 5% CO2 s in the cell culture medium.