DOTAP Transfection Reagent From Roche

DOTAP is a cationic liposomal reagent for transfection (lipofection) of eukaryotic cells with DNA or RNA; I have even heard about researchers using this reagent to introduce new proteins into a cell. The reagent has been on the market for a long time, but nevertheless, it delivers very reliable results.

The day before transfection, one should subculture the cells following standard procedures and seed them into a new multi-well dish or flask at the appropriate cell concentration. I usually use 6-well plates in which I have seeded 4.8 x 10⁶ adherent cells/well fed with 3 ml DME medium containing FCS. The day of the transfection, the DNA/DOTAP solutions are prepared in the following way: 5 µg of DNA are diluted in HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) to a concentration of 0.1 µg/µl in a final volume of 50 µl. For co-transfection with two different DNAs, I use just 2.5 µg DNA each and mix it with HBS buffer in a volume of 50 µl. Another co-transfection scenario is when the selection marker is on a different plasmid. Then you can use, for example, 4 µg of the DNA carrying the gene of interest and 1 µg of the selection plasmid and combine them with the HBS buffer in a 50 µl volume. It is good to use less DNA for the selection marker because then you know that all the cells growing under the selection conditions also carry the gene of interest. In a separate tube, 30 µl DOTAP are mixed with 70 µl HBS buffer. The company states that it is important to use polystyrene or glass tubes for these mixtures, not the typically used reaction tubes made of polypropylene. I always use polystyrene tubes. The DNA/HBS mixture is then combined with the DOTAP/HBS mixture by gently pipetting the solution up and down. Now the transfection mixture is incubated for 10 to 15 minutes at room temperature. Sometimes the reaction becomes cloudy or opaque during this incubation; this shows the formation of the liposome particles. Next, the DNA/DOTAP/HBS mixture is mixed with fresh DME medium. The DOTAP concentration should not exceed 30 µl per ml cell culture medium. I usually use 3 ml medium, which replaces the overnight culture medium form above. I just change the old medium against the DNA/DOTAP/HBS/medium mixture. To achieve a good distribution of the DNA liposomes, I gently mix the cells with the DNA/DOTAP/HBS/medium mixture by slowly swirling the multi-well dish on the bench top of the cell culture hood for about ten times. I also have used the DOTAP reagent for suspension cells. These cells have to be harvested and spun down in a centrifuge to remove the overnight medium. Then, the cells are just gently resuspended in the prepared DNA/DOTAP/HBS/medium mixture before seeding them back in the multi dish or flask. After an overnight incubation of the cells with the DNA liposomes at 37ºC, the medium is replaced one more time and incubated for another 24 h before the selection procedure with the respective selection reagents is started. Alternatively, transient expression can be tested after 48 to 72 h.

I personally have used the DOTAP reagent for stable transfections of mouse L cells, human 293 cells, which are both adherent and really easy to use, as well as mouse P815 cells, which grow in suspension. The respective cell colonies for expression screening were visible after about 2 weeks of culture in selection medium and the whole procedure is highly reliable.