Preparation of cells for transfection (16~24 hours before transfection)

1. Plan what DNAs you would like to transfect. It is recommended to add a positive control, such as a GFP plasmid, to monitor transfection efficiency.

2. Seed the cells at a density of 1.3 x 10⁵ cells per well in a 24-well plates in a total volume of 500µL without antibiotics.

CRITICALIt is important to seed the cells at the recommended density to maximize the transfection efficiency.

On the day of transfection

3. Check the cells are at 60-80% confluency.

4. This protocol is designed for transfecting cells seeded in 24-well plates. You can use this table to adjust the amount of cells and reagents you need for different conditions:

5. Dilute 500ng Cas9 mRNAs in 25µL of Opti-MEM. Mix it gently.

6. Add 50-100ng gRNA into the diluted Cas9 mRNAs. Mix it gently.

7. Add 2µL Lipofectamine RNAiMAX to 25µL of Opti-MEM. Mix it gently and incubate it at RT for 5 min.

CRITICALDo not let the diluted Lipofectamine sit more than 30 minutes.

8. Combine the diluted Cas9-gRNA solution with the diluted Lipofectamine RNAiMAX (total volume: 50µL).

9. Mix it gently and incubate it for 15 min at RT.

10. Add the 50µL of RNA-lipid copmlexes to each well gentally. Mix it gentally by rocking the plate.

Some cells are easy to detach from the plate. It is important to gentally to add the solution in cells to ensure optimla transfection efficiency.

24 hours after transfection

11. Check cells after 24 hours for transfection efficiency.

If you transfect GFP plasmids as a positive control, you can use the percentage of fluoresccent cells to approximate the transfection efficiency.

48-72 hours after transfection

12. Harvest cells for indel analysis.