HeLa transfection... - (Oct/06/2006 )
I've got a "little" problem with my HeLa cells...I'd like to get stable cell line but now, after transfection (CaPO4 method), cells are dying...I wonder where or what I have mistaken...
my protocol is: (DNA+TE+CaPO4) + buffer (vortex), 30 min and then I add this mix to my cells and leave them overnight...the day after I "wash" and change medium
why are they dying???
difficult to say, could be anything from here.
Maybe the plasmid or the content of it is toxic to the cells, are your control cells (e.g. empty plasmid etc...) dying as well?
Maybe decrease the time leaving the transfection stuff on the cells (maybe overnight is too long).
HeLa cells in general are easy to transfect. In case you can get a transfection reagent, try it and compare both outcomes (old reagent and another reagent). As you probably know the cells should be in good growth conditions, let´s say 60-80% confluent at the day of transfection. I used to transfect HeLa cells with siRNA and a buddy of mine with plasmids using HiPerFect from Qiagen. That worked convincing, though I don´t know if HiPerFect is really recommended for plasmid stuff, was just a try...but anyway it worked with high efficiency.
hope this is some input... .
ok thanks for input...
actually, I'm sure about my plasmid, it's not toxic, so I really don't know what to do.
I've thought to change my method but this method has always had good goal...
let's try to change something!
Thanks so much, anyway
Hela's are pretty sensitive to CaPO4, try get your hands on a transfection reagent (LF2000, Fugene, Superfect are found to be pretty good by me and colleagues, but other products might be just as good).