3 replies to this topic

[hogthehedge]

Hi,

Can anyone tell me how to determine how much plasmid DNA to add to 35 mm dishes for a stable transfection?
Someone told me too many nucleotides are cytotoxic to cells. Is this true? What is the reason for their cytotoxicity?
(This person was completely against adding even microgram quantities of DNA, but he was talking about primary cells.)

I'm using Lipofectamine2000, and my cells are fibroblasts, around 70-80% confluent.

Thanks.

[GNANA]

I think it depends on the expressivity of your contruct....some express well and some dnt....ofcourse too much of nucletides are cytotoxic to cells, i dnt know the exact mechanism but i feel other than toxicity induced by nucleic acids the more plasmid you add the more reagents you will be adding (Lipofect in your case), so that will add some more toxicity to cells. whatsoever i wouldnt go beyond 500 ng for 35mm plate..better lesser than 500 ng if you could get good expression in that...

good luck....

[goeselen]

In my lab, we do a "DNA" curve to determine how much DNA of which plasmid gives maximal expression in that particular cell line. Like Gnana said, it depends on the plasmid.

If you don't want to go through all the trouble, your transfection reagent product guide should have some guidelines in terms of how much DNA is optimal for which dish size. Try those first, and if they don't help, you'll most likely need to do a DNA curve.

If you're interested in doing all that work, reply back and I can tell you how our lab does them.

[hogthehedge]

Thanks Gnana and Goeselen,

Goeselen, the curve you mention: is it done by trying out different concentrations and plotting those against transfection efficiency (% cells transfected)?