double transfection plus siRNA - in HEK293 cells (Apr/21/2008 )
I need to transient transfect my HEK293 cells with two plasmids plus an siRNA for biochemical experiments. It shouldn't be that hard (I hope!), but I'm wondering about ratios among DNA and siRNA and concentrations. For a 6cm Petri dish, how much would you use of each?
We are waiting 3-4 days after tranfection with the siRNA experiments, so I think I should use enough DNA to have expression of the proteins after all this time.
What do you think?
Yes, its been done quite often. You need to treat DNA and siRNA as a whole when calculate the ratio of nucleic acid to reagent. It should be within 5-10 ug total DNA and siRNA per plate, but is reagent dependent. You have to decide the ratio between the target and siRNA.
The knockdown kinetics is siRNA dependent. I had good knockdowns (85-90%) at 32-48 hrs post transfection.