72181
293-Free Transfection Reagent
Animal-free polycationic liposomal transfection reagent optimized for the transfection of HEK293 cells grown in suspension culture.
Quality Level
form
liquid
manufacturer/tradename
Novagen®
storage condition
OK to freeze
technique(s)
transfection: suitable
shipped in
wet ice
storage temp.
2-8°C
Related Categories
General description
Features and Benefits
- Excellent transfection efficiency for HEK293 suspension cultures for maximize protein expression levels
- Derived from non-animal sources to move faster from research to development
- Compatible with both serum-containing and serum-free media that simplifies protocol by eliminating media changes
- Minimal cellular toxicity leads to higher protein expression levels
Warning
Other Notes
Legal Information
Signal Word
Danger
Hazard Statements
Precautionary Statements
Hazard Classifications
Eye Irrit. 2 - Flam. Liq. 2
Storage Class Code
3 - Flammable liquids
WGK
WGK 2
Flash Point(F)
67.1 °F - Information taken from reference works and the literature.
Flash Point(C)
19.5 °C - Information taken from reference works and the literature.
Regulatory Information
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Why do I need to perform optimization? How should I go about doing it?
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.
What is the size limit for plasmid DNA?
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.
Is the quality of DNA important for good transfection?
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.
How long should I leave the transfection reagent on the cells? Do I need to change medium at any time after transfection?
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 2-8h of incubation and replaced with complete growth medium.
Can I use the product in the presence of serum?
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.
Can the DNA transfection reagents be used for co-transfecting plasmids?
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.
Will antibiotics interfere with transfection?
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.
How do I scale my transfection protocol when working with different culture volumes?
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.
How important is the cell density/confluency and cell passage number at the time of transfection?
Transfection of nucleic acids requires cells to be actively dividing. Therefore, the optimum cell density for transfection is generally between 50-80% confluency for adherent cells and 1.0-2.0 x 106 cells/mL for suspension cells. Avoid using extensively passaged cells; use a fresh vial of cells if a sudden drop in transfection efficiency is noticed. If necessary, determine the optimum cell density and passage number range for every new cell line, and keep these parameters constant for all experiments to ensure reproducibility.
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