Transfection of Adeno (Calcium Phosphate)

Primary crude virus preparation

Using Stratagene “Virapack” Calcium Phosphate DNA Transfection Kit.

Day 0

Plate Ad-293 cells at 5e5 per 6 cm dish in complete DMEM with serum.

Day 1

Preparation of Adenovirus precipitate:

Add 5 ug of PacI digested pAdE DNA in 450 ul sterile water in polystyrene tube (clear)

Add 50 ul solution #1 and 500 ul solution #2. Gently mix by tapping.

Incubate for 10-20 minutes at RT.

While ppt is forming, wash cells once with serum free media. Do this very gently so you don’t dislodge cells. Leave at RT or without media for as short as possible or cells will lift off plates.

Replace media with 5 ml DMEM supplemented with 6% Modified Bovine Serum (MBS from Stratagene) and antibiotics.

Gently add complexes directly to dishes with swirling motion.

After 3 hours, remove media and ppt completely, refeed with new media.

Day 3

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

Day 6-12

When >80% CPE, harvest media and cells as crude viral supernatant. Freeze/thaw dry ice/24 C x 4 and spin out debris.

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

For adenoviral amplification, can use twice as many cells or let grow 48 hours prior to infection.