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 cm2)

1 x 105

500 ul

 

6 well plate (35mm, 10 cm2)

3 x 105

2 ml

300 ul

6 cm dish (20 cm2)

5 x 105

5 ml

0.5 ml

10 cm dish (60 cm2)

2 x 106

12 ml

1 ml

15 cm dish (135 cm2)

1 x 107

25 ml

2 ml