Difference between revisions of "Fugene Transfection of 293T/COS Cells"

Revision as of 15:28, 15 July 2009

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. Incubat 5-20 min.
7. Split confluent cells 2-3X into fresh dishes as follows:
   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 25 mL COS/FBS
   4. Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
8. Add DNA/Fugene/DMEM to cells
9. Leave mixture on Cells for 24-48h to allow protein to accumulate