Fugene Transfection Protocol

Day before transfection split cells to 60mm dish in a way that on day of transfection the dish would be 60% confluent.

Day of Transfection
1) Add 8ul of Fugene to 100ul of Optimem, avoid touching the the side of the ependorf tube.
2) Incubate 5min at RT.
3) Add combination of DNA ammount depend upon previous expirence usualy 1ug of DNA to Fugene/optimem, and mix well by tapping tube. One can use two different DNA to do cotransfection start with 1:1 ratio.
4)incubate at RT for 15min.
5) Remove media from cells, wash once with optimem. add 4ml of fresh Optimem each dish (60mm dish).
6) Add optimem/DNA/Fugene mixture to cells.
6) Incuabte overnight at 37C.
7) Next day Change Optimem with regular growth media.

Guy