Transfection of J774 using FuGENE 6 reagent

Thursday, October 16, 2003

Description
A method that worked for me, pretty close to the commercial description

Procedure
1 - Preparation of cells for transfection
Day 1:
Plate the cells one-day before the transfection experiment. The appropriate plating density will depend on the growth rate and the condition of the cells. Use cells that are 50-80% confluent on the day of the experiment.
Plating most cell lines at 1-3 x 105 cells in 2 ml in a 35 mm culture dish (or 6-well plate) will achieve this density after overnight incubation.
2 - Preparation of FuGENE 6 Reagent:DNA complex
Day 2:
Use FuGENE 6 Reagent:DNA amounts of 3:2, 3:1, and 6:1 (l and g, respectively) in each well of a 6-well plate or 35 mm culture dish (described below).
FuGENE 6
Reagent:DNA ratio FuGENE 6 Reagent
volume (l) DNA volume
(g)
3:2 3 2
3:1 3 1
6:1 6 1

Preparation of complex
Step 1: In a small sterile tube, add sufficient serum-free medium as diluent for FuGENE 6 Transfection Reagent, to a total volume of 100 l. Add 3 to 6 l of FuGENE 6 Reagent directly into this medium. Tap gently to mix.
The order of addition is critical. The serum-free medium must be pipetted into the tube first.
NOTE: To avoid adversely affecting transfection efficiency, do not allow undiluted FuGENE 6 Reagent to come in contact with plastic surfaces other than the pipette tip.
Step 2: Add 1-2 g DNA solution (0.02-2.0 g/l) to the prediluted FuGENE 6 Reagent from Step 1. Use a total volume of DNA solution between 0.5-50 l.
Step 3: Gently tap the tube to mix the contents. DO NOT VORTEX.
Incubate for a minimum of 15 minutes at room temperature. Continued incubation for up to 45 minutes (for some cell lines up to 2 hours) will not affect the transfection efficiency in most cell types.
Transfection procedure
Step 4: Dropwise, add the complex mixture from Step 3 to your cells, distributing it around the well. Swirl the wells or flasks to ensure even dispersal.
Step 5: Return the cells to the incubator until the time of the reporter gene assay.
NOTE: There is no need to remove the reagent:DNA complex from the cells prior to the reporter gene assay.
If you observe cytotoxicity with the FuGENE 6:DNA complex, refer to section 5.1, Troubleshooting.
For stable transfection experiments, the complex containing medium can be left unchanged until the cells need to be fed.

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Transfection of J774 using FuGENE 6 reagent