Recently I am performing the transfection assay using Fugene 6 reagent in HEK 293T cell. The plasmid includes the EGFP gene. Even I use the vector including only GFP without the fusion gene, the transfection efficiency is very low(about 15%). My plasmid may be well, for the A260/A280 ratio is 1.82.
In 6-cm-dish, when the confluence of 293T reachs 50%,I conduct the transfection. Before the experiment, I place the Opti-MEM at room temperature for 10min and vortex the Fugene 6 to make it evenly and place it at room temperature for 5min. And I mix 200ul Opti-MEM with 12ul Fugene 6 without touching the tube and incubate it at room termperature for 5min. Then I add 4ug plasmid (aobut 12ul) into the mixture prepared above, mix well by vortex and incubate at room temperature for 15min. I drop wise the mixture to the medium evenly. After 48h, I observed very low GFP fluorescence by fluorescent microscope.
Please give me some advice and thank you very much in advance.
Dear Andrei,
I will try again as you suggested.
Now I am wondering whether vortexing will be harmful to Fugene 6 itself. Because I vortex the mixture of Opti-MEM and Fugene 6 as well as Fugene 6 itself.
Thank you very much.
Best
Zhiyuan
Dear Jonas，
Thanks very much for your kind advice.
The Opti-MEM used for transfection is serum free. And you suggest that " first add the DNA to the medium THEN add Fugene". After you add the DNA to the medium, do you incubate the mixture for some time, or do you just add Fugene 6 without incubation?
Best
Zhiyuan
Andras, I am sure that the cells are free of mycoplasma and bacterial contamination. I haven't tried transfection in suspension cells. Have you done such trial and what about the efficiency ?
Hi, Andras
I think it is worth doing in suspension cell. I have done siRNA transfection in suspension HeLa using Hiperfect and get high interfering efficiency.
Hi，everyone
I have tried the assay again using different ratio of plasmid/Fugene6 and found that 1ug plasmids with 4ul Fugene 6 worked well. In my opinion, the cells from diffferent labs may be a little different in cell conditions. So it is necessary to optimize the ratio for cells in different labs.
The ratio is definitely an area where optimization should take place - I've rarely heard that basic protocols end up being the most effective ones. You might also look into complex condensers for HEK293 (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) as these can help improve the overall efficiency.
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