I find that Opti-MEM is necessary. 50% efficiency is pretty low. And like the other person said, you have to always use serum free media. and yes, you can incubate up to 6 hrs, because the lipofectamine is toxic to the cells and replace it with SERUM-FREE media.
I do leave them 17 to 18 hours cause then they start dying, but if your cells can stay alive for 24hrs you can try that. The other thing is that u don't want to give the cells the chance to get rid of the new plasmid. Remember, this is transient transfection, and upon cell division the cells will loose the plasmid.
Curtis, on Jun 14 2010, 02:11 AM, said:
- Is Opti-MEM really necessary? Because I use DMEM and I have 50% efficiency.
- After addition of Lipofectamine to Plasmid, do we need to vortex the mixture vigorously? or just pipetting up and down would be enough?
- Do we need to rotate the plate once in a while after transfection?
- Do we need to remove lipofectamine after 4-6 hours post-transfection and add fresh media?
- Is 16 hours post-transfection enough for cell lysis to detect GFP expression by western blot? what is the recommended time?