Protocol for Lipofectamine Transfection in NIH-3T3 Cells
Using Cells Plated in 8-well Plates
1) Mix 0.5ug of DNA in 96 well plate with 25ul OptiMEM and 2ul Plus Reagent;
incubate at RT 15min.
2) In a separate well, mix 25ul OptiMEM with 1ul Lipofectamine; incubate 15 min at RT.
3) Following concurrent incubations above, mix these tubes and incubate another 15min
at RT.
4) During this incubation, wash each well/dish of cells with ~150ul OptiMEM (remove
with pipette, not aspiration), and then add 150ul OptiMEM to each well.
5) Following DNA incubation, add DNA/Plus/Lipofectamine to well and incubate at 37C
for 45min.
6) After incubation, remove media using a pipettor and wash once with fibroblast media
(500 ml DMEM, 50ml FBS, 5ml Pen/Strep/Glu; filtered).
7) Add 500ul fibroblast media to each well/plate and incubate overnight at 37C.
Note: It is important that you not aspirate media when using the small 8-well plates, as it
can dry and/or dislodge the cells.