stable transfection problem - (Aug/17/2007 )

hey everyone,
I ran into a problem recently trying to set up a stable transfction. I transfected my cells with my linearized plasmid. 24 hours after transfection, the cells were almost 100% confluent so I, trypinized the cells and split them into two different flask and added hygromycin and G418 (my second round of transfection). When I went to look at the cells the next day, most of the cells were floating and dead. Anyone have an idea of what is going on?

Thanks,
joe

-veryslowjoe-

QUOTE (veryslowjoe @ Aug 17 2007, 07:01 PM)

let cells become adherent before adding antibiotics;

or better: choose subconfluency which is not going to become confluent the next day; start after ~24h with antibiotics w/o splitting

stable transfection may not succeed if the overexpressed protein destabilizes the cells

there is an own subforum for question to transfection

-The Bearer-