help with stable transfection screen - (Oct/07/2007 )
Hi! I am trying to make a stable transfected cells with Cos-7 cells. After 24 hours of transfection (lipofectamine 2000) in Cos-7 cells, I splited the cells into 100mm dish with G418 (0.8mg/ml) selection medium. Then I waited for a couple of weeks until my control (non-transfected cells) was dead with G418 and my transfected ones started proliferating. I then screen the postive clones by spliting the cells into 96-wells (1 cell/well). However, I stucked in this step. Each time, after I splited cells into 96-wells, cells are always dying after a couple of days incubation with G418 (0.8mg/ml or 0.2mg/ml) even they started proliferating in the first day or two. I repeated it a couple of times and same thing happened. It seems that while the cell is in single and it just could not survive under growth pressure(I lowered my G418 to 0.2mg/ml and raised the serum to 20%, but nothing changed. Cells are pretty happy before I splited into 96-well.) Any advice for this problem? Thanks.
after transfection, split cells such that there are very few cells in the 10cm plate. We transfect in 6 well plate and after splitting it in 1ml, we make 2x 10 cm plates with 20 and 50ul of cells. then we wait for the single colonies to grow. This usually sorts out stably transfected cells.