I added 3 µg plasmid DNA containing the GFP gene to 75 µL optimem and I added 7,5 µL lipofectamine 2000 reagent to 75 µL optimem. I've incubated the plasmid DNA with lipofectamine and optimem at room temperature (total volume of the DNA-lipid complex was more than 300 µL). I transfected the DNA-lipid complex in dermal melanocyte cells and incubated them for 4 days at 37C, 5% CO2. However, only a few cells had taken in the plasmid DNA and showed green fluorescence. I want to optimize the transfection of melanocyte cells by increasing the amount of DNA.
According to the original protocol, DNA(ug) vs Lipo2000(ul) can be 1:0.5~5, our lab use 4ug DNA with 12ul of Lipo2000. in 1x10^6 cells, mixture added into well with cell seeded but medium without antibiotics. DNA-Lipo. mixture containing medium should be replaced with fresh growth medium 4~6 hours after transfection, remember that analysis should be done in 18~48 hours. Please do check the cell viability or health state by observing cells' shape under microscope, low transfection efficiency may be result from either higher cytotoxicity by amount of Lipo2000 added and antibiotics present in the medium.
You can try that. However, according to my experience, 3 ug plasmid is quite enough and you probably will not get a higher efficiency. I think an ideal transfection is dependent on the health status of cells. Try to keep your cells healthy and fresh during the transfection. Also, if your plasmid has a puromycin resistance gene, try selecting your cells using the antibiotic. Hence, you will have a homogenous cell population.
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