stable transfections - antibiotic dose response (Jun/14/2002 )
I got a question about stable transfection, do you choose a dose of antibiotic that kills all your normal cells by 6 days? Or do you choose a dose that enables 10-20% cells to survive after 6 days- these will be false positives?
You have to choose a dose that will kill all your cells, to be sure that after transfection, only those expressing the resistance will survive. The number of days will also depend on the antibiotic you use. Some take 3 weeks, others will take 5 days. You don't want to destroy them all...
Thanks for the replies. Do you by any chance know how long it will take for Geneticin (G418) to kill all normal cells?
From my experiments, I would say it takes about 15 days to kill al your cells (depending on the number of cells at the start) with G418.
Me too, it takes two weeks.
I am also trying stable transfections and I was wondering which would be a better choice of selection, co transfection of a puromycin resistance plasmid along with the plasmid of my interest (which enables faster selection, in 2-3 days) or work with the neomycin gene that is a part of the vector construct.
CAN ANYONE GUIDE ME AS TO WHAT CELL NUMBER SHOLUD I START WITH FOR TESTING THE DOSE OF THE ANTIBIOTIC?
I use the PcDNA 3.1 to transfect the Mc 3T3 E1 osteoblast cell line. before I transfected the cell, I make the kill curve to decide the dose of G418. from the kill curve, I found that 400 to 500 micro gram per ml is suitable. but, the result is that there is very very small cell died while 800 micro gram per ml kill more cells. Anybody knows the reasons?
I just found this thread and wanted to ask if you know how long it would take for Hygromycin to kill all cells?
dose-response and time-response depend on type of cells; we kill all control cells after 4 days with G418; there is an after-burn effect: you can discontinue antibiotics even if ~5% control cells survived; in our case, they are dead the next day...