I'm in the process of optimizing plasmid transfection to human K562 cells but not very happy with the success rate so far. Any tips or tricks here? Have you tried whether it is better to have the cells in constant shaking during the transfection (as recommended in some protocols) or not?
One thing is, I have used miniprep plasmids (isolated using the Sigma GenElute miniprep kit), as the cell line is highly insensitive to endotoxin. But could this still be a problem for transfection efficiency?
Hi Mikael, it is always better to have a very pure, endotoxin free plasmid DNA and with a majority of supercoiled form. So the purification kit is indeed important. I recommend to optimize your transfection conditions with a reporter gene encoded plasmid which are transfection grade. Additionally, the transfection reagent is also critical and I will suggest DreamFect for these cells.
Best Regards and Good luck
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