What make a plasmid candidate for stable transfection - (Oct/28/2015 )
I'm trying to make stable transfection with plasmids bought from addgene (photoactivatable and mito dendra for photoconvertible). i get only transient transfection. (worked with different cell line and different transfection protocols)
after a while cell lose fluorescence bur continue grow in the presence of the selection antibiotic (G418)
Can someone explain me what happened to the cells?
what make one plasmid able to go stable transfection and another just good for transient transfection?
There are a number of different things that could be going wrong here.
Make sure the G418 is titrated for your cell line, each cell line has different susceptibilities to each antibiotic. Do a kill curve.
The insertion site could be being methylated - the transfected line is still resistant, but the gene of interest is being inactivated.
The protein being produced might be toxic long term (for one cell line).