when to start hygromycin selction - (Jul/02/2007 )

Hi,
I'm traying to do a stable selection with hygromycin. My first attempt, I added hygromycing 24 hours after the transfection. After about one week it seems that all my cells are dead? I'm using 100ug/mL which i got doing a kill curve. Am I adding the hygromycing to soon after transfection? If so how long after transfection should I add hygromycin?

Thanks,
joe

-veryslowjoe-

QUOTE (veryslowjoe @ Jul 2 2007, 03:31 PM)

I would add the selection antibiotic 48-72 hrs after transfection, since thats when the antibiotic resistance protein will be highly expressed. If the gene is only beginning to get expressed and if you add the antibiotics, it will kill the cells.

-scolix-

I have created stable cell lines with hygromycin selection. 24 hours after transfection, wash and add fresh medium to the cells. 48 hours after transfection split cells into fresh media so that they are no more than 25% confluent. Once the cells have attached (2-5 hours) replace media with selection media. Change the media every 3-4 days until foci can be identified.

-rkay447-

from the experience of my boss, it seems that hygromycin solutions are different in terms of efficiency from a stock to an other....
He told me that as hygromycin concentration in the medium should be adjusted fine, you may do a killing curve first each time you prepare fresh solution or buy a new solution.

-fred_33-

Thanks for the advise everyone

Joe

-veryslowjoe-