Thanks for your prompt reply. As I have discussed in my previous post, I have used lipofectamine too with not much luck. I must admit that I have not tried using Fugene. I am working on silencing transporters and reductases. My goal is to establish stable cell line as I have to do some arsenic based experiments on these cell lines. Using kits like lipofectamine would not be ideal for stables. I have tried shRNA DNA based construct. When I do selection with the antibiotic everything disappears indicating there was nothing inside the cells. Now you might ask how do you know the conc. of antibiotic was not toxic? I had done a killing curve and besides using the calculated conc. that was best for selection I have also lowered the concentration to see whether that changes anything. But no luck.
What is the transfection efficiency with fugene6? If you dont mind can you share your protocol for transfection?
As the transfection method for stable (shRNA:
NA based) cell line did not work well, I had to revert back to siRNA and for that again I need somebody with a successful experience. Have you any experience working with HepG2 transfection reagent (Altogen)?
Again I an interested in stable cell line and for that I have all the shRNA's sitting in plasmids ready to go. I have wasted too much time trying to optimize nucleofection kit from Amaxa with only 8-10% efficiency even with GFP. Do you have any suggestions as to what other factors might be affecting this efficiency?
Right now I am attempting to use Retro viral based approach. I am not happy with even this approach. So any tip from your side would be valuable for me.
I appreciate your efforts.
Thanks a ton.
Originally Posted by oBWhat
i have worked with Hepg2 cells for over 5 years. They are solid as a rock, and actually quite easy to transfect.
Fugene6 worked wonders, but also lipofectamine worked well.
How are you transfecting your cells exactly and what are you transfecting (size, amount)? Have you tried an siRNA DNA based construct?