Transfection efficiencies for PolyFect Reagent and 2 lipid-based reagents were compared. Cells cultured in 6-well plates were transfected with a beta-galactosidase-reporter plasmid. The appropriate protocol was used for transfection with PolyFect Reagent. Optimized protocols were used for transfection with Reagents L2 and F; these were: 10 µl Reagent L2 with 3 µg DNA and 15 µl Reagent F with 2 µg DNA for CHO cells, and 10 µl Reagent L2 with 2 µg DNA and 7 µl Reagent F with 3 µg DNA for 293 cells. All transfections were performed in triplicate. Two independent transfection experiments are shown for 293 cells.
The transfection procedure is fast and simple - just add PolyFect Reagent to the DNA solution, mix, incubate for 5-10 minutes, add growth medium (which can contain serum and antibiotics), and pipet the PolyFect-DNA complexes onto the cells. The cells are then incubated for gene expression.
Note the highly branched structure of an activated dendrimer in this schematic representation.
A model of the PolyFect-DNA complex shows PolyFect Reagent (purple balls) interacting with DNA (black) to form a ring-like (toroid-like) structure. The upper right section of the illustration shows naked DNA, the lower section shows the interaction between dendrimers and DNA inside the complex, and the upper left section shows the final complete coverage of DNA within the complex.
Expression of [A] beta-galactosidase and [B] green fluorescent protein (GFP) in HeLa cells is shown. Cells were cotransfected in 6-well plates with beta-galactosidase- and GFP-reporter plasmids using PolyFect Transfection Reagent and the HeLa cell protocol. Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection.