Transfection of Adeno/AAV (Lipofection)

Primary crude virus preparation

Day 0

Plate Ad-293 cells at 3e5 per well in 6 well plate (adeno) or 5e5 per 6 cm dish (AAV)

Day 1

Replace media

Adenovirus

A. 1 ug of PacI digested pAdE construct in 250 ul OPTI-MEM without serum

B. 3 ul of Lipofectamine 2000 in 250 ul OPTI-MEM without serum, incubate 5 minutes at RT

Gently mix A and B together and incubate for 20 minutes at RT. May be slightly cloudy.

Add complexes directly to a single well of six well plate.

48 hours post-transfection, trypisinize each well and transfer to a 10 cm plate.

Replace media every 2-3 days until 80% CPE

AAV

A. 1.5 ug of ITR AAV vector construct and 4.5 ug of packaging vector (DF1-6) in 500 ul OPTI-MEM without serum. (3:1 ITR:packaging vector ratio)

B. 15 ul of Lipofectamine 2000 in 500 ul OPTI-MEM without serum, incubate 5 minutes at RT

Gently mix A and B together and incubate for 20 minutes at RT. May be slightly cloudy.

Add complexes directly to a single 6 cm dish.

48 hours post-transfection, check for fluorescence and harvest cells with cell scraper. 80% of virus will be in cell pellet.

Size of culture vessel (surface area)	Approximate cells/plate for transfection	Volume of media/well	Volume of trypsin
24 well plate (2 cm^2)	1 x 10^5	500 ul
6 well plate (35mm, 10 cm^2)	3 x 10^5	2 ml	300 ul
6 cm dish (20 cm^2)	5 x 10^5	5 ml	0.5 ml
10 cm dish (60 cm^2)	2 x 10^6	12 ml	1 ml
15 cm dish (135 cm^2)	1 x 10^7	25 ml	2 ml