triple-plasmids transfection - (Apr/11/2007 )

I am packaging two pseudotyped retrovirues: HIV-GFP-VSVG, MLV-GFP-VSVG. I used the 293T cells as the target, my transfection reagent is Lipofectamine 2000 (Invitrogen). I followed the manual of Invitrogen. I detected the GFP expression level by the fluorescent microscope, many times I could not see the green fluorescence(exactly just some green plots shown ). I also did the serially dilution of plasmids-DNA, optimizing the transfection efficiency. But I fealured.
So anyone can help me? Or give me a better protocol.

thanks.

I suggest that you use a fixed amount of plamsid DNA and change the lipofectamine 2000 to DNA ratio.
Do it in small well plates for optimization, then move up by multipling by a factor for the difference of surface areas for wells and dishes.
Do it with EGFP expression plamsid first, then do the combinantion.
You can follow the protocol provided by Invitrogen for the amount of plasmid and lipid to DNA ratio as a basis.
Be careful to use high cell density (80-90%, but not confluent) to reduce toxicity, as recommended by the manufacturer.

Try to transfect cells in suspension. I have better efficiencies with it.

But other than this, you have to optimise other factors itself.

I follow the invitrogen protocol. I use 36ul of lipofectamine for a 10cm plate and use 12ug of DNA. If I do triple DNA transfection, I use 4ug of each plasmid.