Fugene Transfection of 293T/COS Cells

Materials

• Fugene-6(Roche cat#)
• OptiMEM or DMEM without serum
• Cells (log-phase growing)
• DNA - typically transfect 50-1000 ng DNA per well

Protocol

1. Warm OptiMEM, COS-FBS Media and PBS -/-.
2. Calculate amount of Fugene needed.
   1. Per ug of DNA need 3 uL Fugene.
   2. Per uL of Fugene need 16 uL of OptiMEM.
3. Add Fugene to OptiMEM, incubate ~5 min.
4. Add required amount of DNA to eppendorf tubes.
5. Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
6. Incubate 5-20 min. Split cells while waiting
7. Split cells to desired density. Cells double about in ~24h so for confluent cells the next days split 1:2. For immunofluoresence split 1:4 from a confluent dish. If cells are subconfluent split to a higher density:
   1. Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
   2. Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
   3. Add 24-48 mL COS/FBS depending on density (24 mL to split 1:2, 48 mL to split 1:4)
   4. Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well). Place sterile coverslips in wells if immunofluoresence is required.
8. Add DNA/Fugene/DMEM to cells.
9. Leave mixture on Cells for 24-48h to allow protein to accumulate.