I use the calcium phosphate method of transfection and the majority of the cells are GFP positive after less than 24 hours. I have also used PEI and liposomes. I tried once a commercial transfection reagent and the efficiency was very poor.

Are your stocks of plamid old? After a couple of years these will produce lower titers, whether in a freezing buffer or glycerol stock. Also, are you going through 4 complete freezing/thawing cycles? We work extensively with multiple AAV serotypes and after 2 each of freezing/thawing processes we notice appreciable drops in the virus yield.

quality of 293T cells and their passage number is also very important.

Looks like you used the same amount of DNA for all 3 plasmids. Try the ratio of 2:1:1 for AAV GFP, HELPER and RC2.

Thank you very much. I am going to try out these things and come back to you guys.

All of the other tips are good. When I used to make AAV in 293 cells, I found the timing of transfection was very important. I would get good efficiency if I xfected between 12-16 hrs post seeding, but it would drop off after that window. The other thing that caused lower xfection efficiencies for us was not the age of our DNA, but the quality of the prep. If the level of salts in your DNA prep are too high, you could be getting poor xfections. We found that the qiagen kits worked really well, but we had to reprecipitate our DNA from some other kits to get good results. We also found PEI worked the best.

We are using CaPo or DreamFect Gold with sucess. Important parameters as described above are cells heatlh, exponential growith, DNA quality and purity and in your case optimizeing the ratios among the plasmid.