|Cell Lines:||Primary Cells:|
| Aortic smooth muscle cells|
67.1 °F - Information taken from reference works and the literature.
19.5 °C - Information taken from reference works and the literature.
Each cell type behaves differently, by carrying out an optimization, the best transfection condition for your particular cell type can be determined. In other words, you can avoid putting too much transfection reagent on your cells, which may cause unnecessary toxicity issue and waste of precious transfection reagent. Optimization is suggested for every new combination of cell type and plasmid. The most important parameters are cell density and ratio of transfection reagent to DNA. Start with the volume of the selected transfection reagent (1x) and plasmid amount (1x) as recommended in the User Protocol. If those conditions do not yield the desired results, an optimization experiment can be performed. In a 24-well plate, plate the same amount of cells in each well. Set up a gradient across the plate and add the appropriate volume of transfection reagent (0.5x, 1x, 1.5x, 2x, 2.5x and 3x). Set up a gradient down the plate and add the appropriate amount of plasmid (0.5x, 1x, 1.5x and 2x). With a reporter gene in the plasmid, the optimal condition can be easily determined.
Large plasmids in the range of 12-15 kb can be transfected. We have cloned and expressed inserts encoding large proteins (including β-gal) without difficulty in mammalian cell lines.
Yes, it is essential that the DNA to be transfected is of high quality and free of endotoxins. Plasmid DNA preparations should include an endotoxin removal step.
Since our nucleic acid transfection reagents are compatible with serum-containing media, medium change after transfection is not necessary. The majority of cell types can be incubated with the transfection mix for 24-72h without any media change, and then harvested for the desired downstream application. If media change is necessary due to the toxicity of the protein being expressed, the transfection mixture can be removed after 2-8 h of incubation and replaced with complete growth medium.
Yes. Our nucleic acid transfection reagents are effective for transfecting cells in media with or without serum. While cells can be incubated in media containing serum, it is absolutely critical that serum is NOT present during formation of the transfection reagent/DNA complex. For most applications, we recommend adding the transfection reagent/DNA complex (formed in serum-free media) to cells grown in complete growth media. For certain cell lines and experimental conditions, serum starvation of cells might be required. Since serum provides growth factors and nutrients, transfection efficiencies achieved with growth in serum containing media are typically better than those in serum-free media.
Yes. Multiple plasmids can be transfected into the cell at the same time. The key is to maintain the optimal ratio of total DNA (all plasmids). See the User Protocols for more information on the ratio of reagent to DNA.
We do not recommend including antibiotics during the formation of the transfection reagent/DNA complex. Increased cell permeability during transfection causes high antibiotic influx, resulting in cell death. Some antibiotics (such as kanamycin) are cationic and can therefore interfere with transfection. Antibiotics such as penicillin and streptomycin can be present in the complete growth media (with serum) which is used to grow the cells. If you are generating stable transfectants, add selection antibiotics (e.g., G 418 or hygromycin) 48-72h after transfection.
For most standard culture formats, guidelines are provided in the User Protocol. If you are using different culture volumes, vary the amounts of DNA, transfection reagent, cells, and culture media in proportion to the relative surface area while keeping the transfection reagent: DNA ratio constant.
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