PolyFect Transfection Reagent (4x1ml)

Manufactured by QIAGEN, this transfection reagent is intended for optimized transfection of COS-7, NIH/3T3, HeLa, 293 and CHO cells in 6-well plates, with adapted volumes for 60 mm and 100 mm dish formats. Upon arrival the reagent should be stored at 2–8°C. The accompanying Quick-Start Protocol notes that cells must be in optimal physiological condition and reach 40–80% confluency on the day of transfection. Plasmid DNA quality strongly influences transfection efficiency, reproducibility and toxicity, and serum, proteins and antibiotics present during complex formation will significantly decrease transfection efficiency.

For a 6-well plate, the protocol specifies seeding 4 x 10^5 cells per well for COS-7, NIH/3T3, HeLa and CHO cells, and 6 x 10^5 cells per well for 293 cells. DNA dissolved in TE buffer (pH 7 to pH 8), at a minimum concentration of 0.1 µg/µl, is diluted with serum-, protein- and antibiotic-free growth medium to a total volume of 100 µl. For COS-7, NIH/3T3 and CHO cells, 1.5 µg DNA is combined with 10 µl PolyFect® Transfection Reagent; HeLa cells use 1.5 µg DNA with 12 µl reagent; 293 cells use 2 µg DNA with 20 µl reagent. After mixing, samples are incubated for 5–10 minutes at room temperature (20–25°C) to allow complex formation, then added to cells in fresh medium. Table 1 in the protocol provides the corresponding reagent and DNA volumes for 60 mm and 100 mm dishes across the four cell lines.