View source for Fugene Transfection of 293T/COS Cells

==Materials==
* Fugene-6 (Roche cat# 11815091001)
* OptiMEM or DMEM without serum
* Cells (log-phase growing)
* DNA - typically transfect 50-1000 ng DNA per well

==Protocol==
#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.
[[ Category: Cell Culture ]]
[[ Category: Transfection ]]