DNA delivery into cultured cells

Lipofectamine 2000 (Life Tech) was used to deliver gRNA plasmids (2.0 μg each) into CAD5 cells according to the manufacturer’s instructions and at a cell culture plate confluency of approximately 60–70%. 48 hours post transfection, cells were harvested and processed for GFP-FACS sorting. DNA delivery into MEFs by nucleofection was optimized using manufacturer’s instructions and the Lonza-MEF starter Kit-VPD-1006 and Amaxa 2b-Nucleofector device. MEF solution-2 and T-20 program were selected for optimal nucleofection. MEFs were grown in T25 flasks to a confluency of 60–70%. Two times 10⁶ cells per nucleofection were collected at 250 × g for 5 minutes and resuspended into pre-warmed S1-supplemented MEF solution-2. gRNA plasmids (each 2.0 µg) were added to the cells and incubated for 10 min on ice. The cells with DNA were transferred to a 100 µl nucleocuvette and electroporated using T-20 Nucleofector 2b program. Post pulse, 500 µl of pre-warmed growth media was added to the nucleocuvette and the cells were transferred to a 12 well plate. Forty-eight hours post nucleofection, cells were harvested and processed for GFP-FACS sorting.