concentration of DNA for stable transfection with lipofectamine 2000 and the per - (Aug/09/2007 )

hi
I am about to start my stable transfections with lipofectamine 2000. I wondering how much DNA I should use for stable transfection with lipofectamine 2000 tranfection reagent.What percentage confluency should be used for stable transfection? Do any one has protocol for stable transfection with lipofectamine 2000. For selection G418 will be used.
Thanks in advance
sister

see protocol here
in 6well plate i use 60% confluency, 1µg DNA and 3µl lipofectamine. (worked on HeLa and Hek293cells)

Thanks fred!!
So you add 1 microgram of DNA and 3 microliter of lipofactamine per well. How much quantity of transfection mix you add to each well in 6 well plate ? when transfection complex is added t o the seeded cells at that time do you add serum and antibiotics in medium?After transfection after howmany days you transfer to 10 cm plates and how. I am sorry, in one reply asking so many question. Hope you would not mind!!
thanks
sister

I add 1 microgram of DNA and 3 microliter of lipofectamine per well. They have been diluted eah in 200µL of serum free medium. i add the 400µl over the cells and complete to 1ml with SFM. 5h lat'er i add 1ml of 20%serum medium without antibiotics. The day after i replace with frehsh medium. I start selection on day 3 and transfert by tripsinization when required (confluency)

For transfer of cells from 6 well plate to 10 cm plate you use trypsin. Do you centrifuge cells after additing trypsin?. How much trypsin you use per well.Can you please explain me the procedure in detail.thanks
sister

ok i czn explain. But i think honnestly someone in your lab should tell you. If you don't do it in your lab, at least ask someone to look the procedure before doing it yourself alone. It's far better.

I use 0.5ml of trypsin solution per well and add 0.5ml of PBS per well.
I incubate 5' at 37° for 293 or hela cells (but it may varies amongst cell line types).
Then i add 2ml of medium,collect the cells (i eventually pipett up and down to fnalize cell detachment and reduce clums), transfert the cels in 15ml tube and spin 250g 2'.

you can't use PBS. It detach cells (see the thread on it) and does not contains the nutriments needed for cell survival. My eficiency growed with optimem, but for stable transfection you can use our medium without serum and without antibiotics during the time transfection mixture is applied on the cells (ranging so from 5h to 24h).
You don't have to spin your cells right after transfection, except in the case it's suspension culture and you want to get rid of transfection mix.

in your lab, if you do cell culture it's the same as standard transient tranfection, and for cel spinning it doesn't harm thm. If you're worried, spin at 200g for 5', that's gentlier