I am using FugeneHD and wish to start a stable transfection using HeLa cell lines. As a starting point, what would be a good concentration of plasmid to transfect HeLa cells with?
I have read the manual. From your experience, do you have a recommended concentration for stable transfection?
Chances of a stable transfection are pretty low anyway. You could just perform a normal transfection (Say using 500ng or 100ng). You will still have to perform a serial dilution/limiting dilution to obtain individual stably transfected clones.
Sanath is correct. Optimise your transfection protocol with a marker plasmid (e.g. luciferase or GFP under a viral promoter) as outlined in the manual, then use the best transient condition you found for your plasmid (I use 200.000 cells per single well in 6-well plate), add the selection antibiotic and wait for (most of the) cells to die. It can be that you have very few survivors and they may need weeks (!) to start proliferating. Scratch the surviving cells off and do another round of selection. From the surviving clones you may try a single clone selection. I don`t do that and use the "mix" clones.
If you are specifically looking for stable transfection, using a lentiviral packaging system will be much more efficient and likely to yield results than using a transfection reagent which will likely lead to a transient transfection.
Kalkal is correct, viral infection is much more efficient for stable transfection. However, in Germany you need a gen tech lab security level 2 to be allowed to do so.
Thank you all for your input and advice. I will not be able to carry out the lentiviral packaging system. I have to perform a serial dilution to obtain individual stably transfected clones.
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