1. Add 8ml growth medium to dish (not flask); warm 30 min in incubator.
2. Prepare nucleofection solution:
- 19 ul supplement
- 81 ul solution L
let sit at room temperature.
3. Prepare the flask: wash with PBS—and trypsinize. Add 1 pipette full of medium, place cells in 15ml conical tube and spin, 2 centrifuge, setting 1 for 10 min.
4. Remove as much media from above the pellet as possible. Add 2ug DNA and 100ul nucleofection solution. Using the p1000 (want large pipette tip) pipette to resuspend the cell pellet. Add this solution to the cuvette.
5. Put in Amaxa and run program X-001. Use a transfer pipette to add media from the cuvette to the dish. Put dish in incubator.