915
edits
Changes
updated protocol
==Materials==
* Fugene-6(Roche cat#)
* 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 -/-.
#Add required amount of DNA to eppendorf tubes.
#Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
#Incubat 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 1:2-3X into fresh dishes as follows. 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 25 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 ]]
