help ! selecting transfected colonies - (Aug/27/2006 )

Dear transfectors!!

I am trying to determine the best dose of hygromycin that i can use to select my transfected colonies. (working with chinese hamster cell lines). I have tried several concentrations (from 50-250 microgram per ml) on my host cells and changing the media plus antibiotic every 1-2 days. Each time the media has turned yellow and lots of dead cells but still surviving colonies can be seen. How could we know if the cell death is because of the overgrowth of the culture and consequent bad condition or because of the antibiotic?

hygromycin is selection is not clean. You will see some background. Anyhow try uping the dose to 400 micrograms per ml. I know the stuff is expensive but give it a try. I am worried about the media turning yellow though. No bacteria, mycobacteria or anything contaminating your cultures, i hope.

P.S: Are your hamster cell, CHO cells?