d. Nucleofect RNP complex and donor plasmid into recipient cells using Amaxa 4D-nucleofector (Lonza). The choice of nucleofection program and solution is cell line-dependent and requires optimization beforehand. For commonly used cell lines, optimized nucleofection conditions are found on the Lonza website. The nucleofection programs for cell types we have successfully used in the lab are provided in the materials and equipment section.
i. Mix 16.4 mL cell line specific nucleofection buffer (e.g., P3 Primary Cell) with 3.6 mL buffer supplement.
ii. Add 500 ng pUC19 donor plasmid (in the smallest volume possible) to 20 µL nucleofection buffer mix.
iii. Resuspend 2x10^5 cells in nucleofection buffer/plasmid mix.
iv. Transfer cell suspension to tube with assembled RNP complex and mix solutions by carefully pipetting up and down with a P200 pipette tip. v. Transfer nucleofection mix into 16-well Nucleocuvette strip.