▲ CRITICAL STEP Do not plate more cells than the recommended density, as doing so may reduce transfection efficiency. 9On the day of transfection, cells are optimal at 70–90% confluency. Cells can be transfected with Lipofectamine 2000 or the Amaxa SF cell line 4D-Nucleofector X kit according to the manufacturers’ instructions. Transfections should be performed as follows: for sgRNAs cloned into pSpCas9(BB), transfect 500 ng of sequence-verified CRISPR plasmid (pSpCas9(sgRNA)); if you are transfecting more than one plasmid (Box 2), mix them at equimolar ratios and use no more than 500 ng of total DNA. For sgRNA amplified by PCR, mix the following: pSpCas9 (Cas9 only)400 ngsgRNA amplicon from Step 5A (each)20 ngpUC19 (carrier DNA)Fill up total DNA to 500 ng
