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

1. Cell preparation:
Plate 5E+6 AAV·HT™ packaging cell (or other HEK293 cells) to a 150mm tissue culture dishes in 15 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 (per 150 mm plate):
A. DNA dilution: the plasmid quantities for transfection are Capsid 10 ug, pHelper 16 ug, cis-plasmid 10 ug, diluted with 0.5 ml basic DMEM media (serum and antibiotics free) followed by gentle mixing.
B. GenJet Plus (Cat # SL100499) dilution: dilute 90 ul GenJet plus reagent into 0.5 ml basic DMEM media (serum and antibiotics free) followed by gentle mixing.
C. Put "B" in in "A" and mix well, stay at room temperature for ~10 minutes.
D. Add the mixture from "C" to a 150 mm plate drop wisely.

3. Cell Harvest:
Harvest cell 48 to 36 hours after transfection. Yield is around 1E+13 VG total from 10x150 mm plate.