▲ CRITICAL STEP We recommend transfecting in technical triplicates for reliable quantification, and including transfection controls (e.g., GFP plasmid) to monitor transfection efficiency. pSpCas9(sgRNA)-2A-GFP or pSpCas9(sgRNA)-2A-Puro may be used in place of pSpCas9 if fluorescence sorting or drug selection, respectively, is desired. In addition, the pSpCas9(BB) cloning plasmid and/or the sgRNA amplicon may be transfected alone as a negative control for downstream functional assays. 10Add Lipofectamine complex to the cells gently, as HEK 293FT cells can detach easily from the plate, which will result in a lower transfection efficiency.11Check cells after 24 h for transfection efficiency. The percentage of fluorescent cells in the transfection control (e.g., GFP) can be estimated by using a fluorescence microscope. Typically, more than 70% of cells are transfected.
