293T cells death after transfection

[Scientist in crisis]

Hi everyone!

I'm new in Molecular Biology and my first task was to do a co-transfection of 293T cells using a LP-IRES2-EGFP vector and a pRSV-Rev and the using Lipofectamine 2000 and Opti Mem with a lot of bad results. First, I had a very poor transfection efficiency until we realize that cells had more than 60 passages, so we change cells and the efficiency get better (around 40%), but my cells start to die, a lot. I change medium after 6 hours of transfection and harvest the cells 24 hours. I also use about 90% of confluency and a DNA - Lipofectamine ratio of 2.5. I'm in crisis. Someone has some idea of whta is happening?

Thanks,
:

[eran.hadas]

Hi
No need to use lipofectamine. We been using linear 25 KD polyethyleneimine at 6:1 to DNA for several years now to produce different viruses from transfection 293T with very good results.