Transfection protocol of plasmids for production of ultra-high titer Adenovirus

1. Cell preparation:
Plate 5E+4 per well of Ad·MAX™ 293 cell (or other HEK293 cells) to 24 well plate 0.5 ml DMEM media containing 10 % FBS. Make sure the cells are distributed evenly. Place the plate in an incubator in 5% CO2 in air and allow cells to grow to more than 90% confluence (24 to 48 h.). Feed cells with fresh media before transfection.

2. Transfection procedures:
A. DNA dilution: dilute linearized Adenovirus DNA 1ug in 50 ul basic DMEM media (serum and antibiotics free). Mix gently.
B. GenJet plus dilution: dilute 3 ul GenJet plus (Cat. No. SL100499) in 50 ul basic DMEM media (serum and antibiotics free). Mix gently.
C. Put "B" in in "A" and mix well, stay at room temperature for 10 to 20 minutes.
D. put the mixture from "C" in tissue culture dishes drop wsiely.

3. Cell Harvest: harvest cell around 10 days after transfection, feed cells with fresh media at 5th days post transfection, yield is from 5E+9 to 2E+10 total PFU.

4. For the 6 well plate, plate 2E+5 cells per well. dilute 3 ug linearized adeno DNA in 0.125 ml basic DMEM media (serum and antibiotics free). Mix gently. Dilute 9.0 ul GenJet plus (Cat. No.L100499) in 0.125 ml basic DMEM media (serum and antibiotics free), mix gently. Follow the above transfection procedures.