Alternatively, if you are using ssODNs, simply resuspend them to a final concentration of 10 μM (see Step 4) and skip Steps 15 and 16. 15Run a small amount of the linearized plasmid alongside uncut plasmid on a 0.8–1% (wt/vol) agarose gel to check for successful linearization. Linearized plasmids should run above the supercoiled plasmid.16Purify the linearized plasmid with the QIAQuick PCR Purification kit, and elute in 35 μl of EB buffer.17Preparation of cells for transfection. Culture HEK 293FT in T75 or T225 flasks. Plan ahead to have sufficient cells for the day of transfection (2 × 105 cells per transfection if you are using the Amaxa SF cell line 4D-Nucleofector X kit S).18Prewarming plates for transfection. Add 1 ml of warm D10 medium into each well of a 12-well plate. Place the plates in the incubator to keep the medium warm.19Use option A in the table below for preparing the co-transfection of the HDR targeting plasmid with the Cas9 plasmid or option B for the co-transfection of ssODN with the Cas9 plasmid. To prepare transfection controls, see Step 9.
