Hi every one....
I am looking for some valuable tips to achieve stable transfection in my cells. I have transfected the cells (using lipofectamine 3000) 36 hours ago and as I have read some protocols for stable transfection, I have to add selection antibiotic to my cells at 48 hrs post transfection. But I am worried as my cells are around 90% confluent and selection atbiotic at such confluence won't be effective in killing nontransfected cells.
Can I passage my cells at this instant and then add selection antibiotic next day or I should passage the cells in selction media at this instant?
Your suggections will be of great help
Thanking you!
It will take some time, I would let them sit. Allow for the selection to happen.
I would wait to subculture until outgrowth is observed with antibiotic (G418) in medium. I attached some of my protocols.
Best Wishes!
I see no reason why you wouldn't be able to transfer the cells to a larger flask in antibiotic containing medium. The cells that are transfected would be resistant. I have done that before and it works fine.
Thanks Anderson, I passaged the cells and after 12 hours I added the antibiotic to them and hope it works.....
@ Ozkan, I was intending to passage the cells first and then add selection media after 12 hrs because of the fact that it is not necessary that your transfected plasmid starts expressing at 48 hr post transfection, mostly it takes about 72 hrs as well.... Since my cells were getting overconfluent at 48 hr, that is why I was intending to passage them forst and add antibiotic at around 72 hrs P.T. this way making it more sure that if transfection has occured, the protein will be expressing at this instant which will save cells against antibiotic!
And many thanks, anyways...
- Badges
- Science method