Fugene Transfection of 293T/COS Cells

Protocol

1. Warm OptiMEM, COS-FBS Media and PBS -/-.
2. Calculate amount of Fugene needed.

• Per ug of DNA need 3 uL Fugene.
• Per uL of Fugene need 16 uL of OptiMEM.

1. Add Fugene to OptiMEM, incubate ~5 min.
2. Add required amount of DNA to eppendorf tubes.
3. Add 51 uL/ug OptiMEM/Fugene to each DNA tube.
4. Incubat 5-20 min.
5. Split confluent cells 2-3X into fresh dishes as follows:
6. Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
7. Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
8. Add 25 mL COS/FBS
9. Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
10. Add DNA/Fugene/DMEM to cells
11. Leave mixture on Cells for 24-48h to allow protein to accumulate