have seeded 110000 cells in a 24 well plate and transfect cells with 1 migrogram plasmid + 2.5 microlitre of Turbofect. The GFP flourescent as wells as normal picture after 48 hour is attached here. Would you pleas help me how can I get better transfection efficiency?
I haven't worked with turbofect, but this one (https://altogen.com/product/mcf-7-transfection-reagent-breast-cancer-htb-22/) we've tested to have around 80% efficiency with siRNA transfection.
Soheila
FYI, your picture is not attached.
Three suggestions:
(1) Start with healthy log phase cells.
(2) Use 10,000 cells per well and transfect 1-8 hours after plating.
(3) Titrate the DNA/Turbofect using a "checker board" strategy usually supplied by the manufacturer in the recommended medium.
Be sure your cells have been pre-adapted to grow in the manufacturers recommended medium.
Michael
You are taking correct cell density for 24 well plate. You can use different ratio of DNA/Turbofect 1:2 , 1:3, 1:4 . We generally use Lipofectamine 2000 for transfection and titrate with similar method.
Micheal,
I have follow the recommended protocol .The only parameter I have changed, is number of cells seeding in the 24-well plate. In stead of 60,000 cells I have seeded 110,000 cell. 60,000 cells is too low for transfection. I transfect 18 hour after seeding.
Aftab,
The concentration of plasmid I have transfected is 300 ng (3.5 μl plasmid) + 2.5 μl transfection reagent. By different ratio of DNA/Turbofect , do you mean the volume ration?
Hi Soheila,
You should take around 1 ug of DNA and 3ul of turbofact. Similarly you can also increase the turbofact to 4 ul for 1 ug DNA. Also please check the quality of plasmid 260/280 it should be 1.8. DNA should be endotoxin free. send the pictures of transfection for further troubleshooting.
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