Transfection of HEK293 cells

Calcium phosphate transfection was used to generate stable cell lines in HEK293 cells. For each 10 cm dish, two separate reaction mixtures were prepared — (a) 0.5 ml of 2× HEPES buffered saline (HBS) (280 nM NaCl, 1.5 mM NaHPO₄·7H₂O, 50 mM HEPES pH 7.0) and (b) transfection mixture containing DNA (10 μg — containing 9 μg of the Flp recombinase vector, pOG44 and 1 μg of the cDNA vector), CaCl₂ (2 M, 61 μl) made up to a total volume of 0.5 ml using sterile H₂O. The transfection mixture was then added drop-wise to the HBS tubes with gentle shaking to ensure sufficient aeration. This was done to make sure that the calcium phosphate-DNA precipitate is as fine as possible as this enhances transfection efficiency. The resulting mixture was then added on top of the cells in a drop-wise manner.