Phenol/Chlorophorm extracted DNA - ok to use for transfection??? (Oct/23/2006 )
One simple question: is it ok to use Phenol/Chlorophorm extracted DNA for my transfection??? Why or why not??? Or do I always have to use DNA cleaned over a column???
I want to transfect A549 cells with my construct btw
yes, it is okay, but only if you make sure that all the phenol is removed. I also do a PEG percipitation after the phenol step. And to finish off, I do at least 2 consecutive resuspension and EtOH percipitation steps to be absolutely sure that any remaining phenol and PEG is removed.
Also it would depend on how sensitive your cells are to endotoxins (whose production rapidly increases near the late log phase/early stationary phase). Hela cells are okay, but phenol/chloroform doesn't remove sufficient endotoxin for more sensitive cells.
EDIT: I haven't worked with A549 cells, and therefore can not advice with regards to endotoxins.
P/C is ok for transfection. I use to do P-P/C-C-C to get the cleanest plasmid. Here in the lab works in a wide variety of cells, like A549, NCI-H69, even ES cells.