Fugene Transfection of 293T/COS Cells
From Bridges Lab Protocols
- Fugene-6 (Roche cat# 11815091001)
- OptiMEM or DMEM without serum
- Cells (log-phase growing)
- DNA - typically transfect 50-1000 ng DNA per well
- Warm OptiMEM, COS-FBS Media and PBS -/-.
- Calculate amount of Fugene needed.
- Per ug of DNA need 3 uL Fugene.
- Per uL of Fugene need 16 uL of OptiMEM.
- Add Fugene to OptiMEM, incubate ~5 min.
- Add required amount of DNA to eppendorf tubes.
- Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
- Incubate 5-20 min. Split cells while waiting
- 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:
- Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
- Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
- Add 24-48 mL COS/FBS depending on density (24 mL to split 1:2, 48 mL to split 1:4)
- Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well). Place sterile coverslips in wells if immunofluoresence is required.
- Add DNA/Fugene/DMEM to cells.
- Leave mixture on Cells for 24-48h to allow protein to accumulate.