Puromycin and HEK293 cells - (Aug/09/2006 )

Hi there!

I have a problem with puromycin selection:

I seeded 300000 HEK293 cells in 1 well of a six well plate and transfected it with 1 µg pPur from Clontech after 24 h. 24 / 48 h after transfection i started selection with 1 µg/ml Puromycin (did a test before to determine necessary concentration to kill untransfected cells). I have done a transfection control with pEGFP-N3 as well, transfection was fine (70 %). After 5 days all the cell were dead, transfected and untransfected cells.

any idea what is wrong? No one in my lab worked with puromyvin before so no help here...

Thanks Stardust

err...
well divide by 2
or your transfection was unsuccesful.
Finally, maybe you only have poor transfection efficiency, and cell clones will appear in 2weeks?...
I encounter same prob, and continue selection to see this possibility

Sometimes, the transfected HEK293cells lose the resistance gene and died in media containing puromycin. I don't know why.

hmmmm transfection efficiency...as i said with pegfp-n3 it was 70 % (in the same experiment...) i have done the transfection 3 times now and always the same problem. Digested plasmid to see if it is maybe not pPur but fragments were ok. maybe the dna preparation is dirty or something? would that be possible?

Thanks for your help!

Stardust