| 1. | Feed the 6 cm plate 3 hours before using. |
| 2. | Wash with HBSS-Ca-Mg. |
| 3. | Repeat wash. |
| 4. | Add 1 ml of 0.25% trypsin to plate. |
| 5. | Incubate at 37°C for 5 minutes. |
| 6. | After trypsinization is complete, add 2 ml ES media. |
| 7. | Transfer the suspension to a 15 ml tube. |
| 8. | Using a transfer pipette, pipette up/down 20 times. |
| 9. | Centrifuge at 1000 RPM for 5 minutes. |
| 10. | Remove supernatant. |
| 11. | Resuspend cells in 3 ml of ES media. |
| 12. | Aliquot 1 ml to 1.5 ml tube for microinjection. Place this tube on ice. |
| 13. | Centrifuge the remaining 2 ml at 1000 RPM for 5 minutes. |
| 14. | Resuspend the cell suspension in 2 ml of 1 X freezing media. |
| 15. | Aliquot into freezing tubes and freeze for later use. |
| 16. | Take the ES cells that are on ice to the GEM Core by 9 a.m. the morning of scheduled injection. |
