Six Well Cell Transfection of 293 Cells

This is a method devised by Marc Diamond: Primarily, this method uses Lipofectamine plus reagent as the vehicle for introducing the plasmid DNA into the cell.



OptiMEM (serum free-media), Gibco brand

Lipofectamine plus reagent which comes packaged together, Gibco brand

293 Cell/Cos cell media (kept at 4°C) we made ourselves

GFP DNA plasmid

One’s own DNA plasmid

Take two polystyrene tubes

Label tubes #1 and #2


Into Tube #1

100 ul OptiMEM

3.2 ul of the plus reagent

0.8 ug GFP

1 ug DNA (your own)

Incubate this mix for 5 min


Into Tube #2

100 ul OptiMEM

2.2 ul Lipofectamine

Add tube #2 to tube #1, incubate for 15 min

At the same time aspirate away cell media from the six well dish and replace with 37°C warm OptiMEM (800 ul per six well dish).

After 15 min add tube 1 and 2 mixture to six well dish, which contains 800 ul OptiMEM solution. Swish solution around a little.

Leave solution in well for 3 h. After 3 h aspirate away Lipofectamine/plus reagent/OptiMEM media and replace with 293 Cell/Cos culture media.

Around 48 h later lyse cells.

Note: Cell Transfection success may lie with the number of cells you begin with on the plate. With a very successful transfection (~50–60%) I started with ~3.2 million cells/dish.