Troubles in transfection methods! - All cells die before making a selection (Nov/03/2008 )

Hi everyone,
I´m new in this forum and in siRNA experiments... so I need some help... I´ve been trying to transfect MT-4 and HELLA cells with lipofectamine 2000 (Invitrogen) and a pSilencer retro vector (Ambion) with the target sequence. But after transfection all cells begin to die, when I am going to make the selection they are almost all dead...
I don´t know what´s happening....
Any ideas???

Thanks,

Diana

Are you using an endotoxin free kit to prepare your DNA?

Lipofectamine can be toxic to cells if left for long time. After 6 hrs of transfection the media needs to be changed.

I changed the media after 4 hrs... and my kit is endotoxin free... Could my plasmid DNA be toxic?

Just observations from my own experience... have you tried transfecting in lower amounts of your vector? Some cell types are more sensitive to foreign DNA. I've no experience with your cell types, but our cells are more than happy with lipofectamine 2000- After 4hrs with it, I don't even change the medium- just top it up with fresh medium and this goes for another 24hr before a complete change.

Also, the other post about endotoxin is a possibility. Our immune cells seem to show some sensitivity to DNA that is not endotoxin-free. That is, I found minimal death with a endotoxin-free maxi prep. of my vector DNA compared to an uncleaned mini prep.

Hi, thanks for your feedback. I did not want to leave the lip for more than 4-5 hrs because I thought that this could be the reason they were dying for... I can try to see what happens.

Thanks :-)