AAV Production Protocol

1. Timeline

- Day1: Cell seeding

- Day2: Cell transfection

- Day5: Cell harvest & lysis

 

 

2. Protocol

 

Day 1     Seed HEK293T cells with DMEM/10%FBS/1%a.a medium at each dish

*  10cm dish: 4.0x106 cells

    15cm dish: 9.0x106 cells

* Confluence at 70-80% next morning

 

Day 2     Proceed transfection as below.

 

* PEI solution (1ug/ul)

- Dissolve ~100mg of branched PEI solution in ~10ml of DDW.

- Dilute at 10ug/ul in DDW and adjust the pH to 7.0 with HCl.

- Filter the solution through a 0.22um membrane, aliquot 0.5-1ml into sterile tubes, and store the tubes at -80℃

* pAAV : pRepCap : pHelper = 1: 1: 2 (molar ratio)

Plasmid	Plasmid Size(bp)	100 mm dish	150 mm dish
pAAV		6 ug	12 ug
pRepCap		6 ug	12 ug
pHelper		12 ug	24 ug
Total bp		24 ug	48 ug

 

- Prepare the plasmid/PEI mixture (1:3 ug ratio).

1) For a 100 mm dish, add all three kinds of plasmid to tube 1 containing 0.5ml PBS, vortex and spin down.

2) Add 72ul of PEI solution (1ug/ul) to tube 2 containing 0.5ml PBS, vortex and spin down.

3) Transfer the solution from tube 1 to tube 2, vortex, spin down, and incubate for 5-10min at RT.

4) Dropwise in the dish while mixing.

 

Day 5     Harvest Virus.

 

* Lysis Buffer

Reagent                                                    Final Concentration

1M Tris-HCl (pH 8.0)                                50 mM

NaCl                                                         150 mM

MgCl2                                                       2 mM

  - Sterilize using a disposable 0.45-µm filtration

  - hood and store at room temperature

 

1) 72 hr after transfection, harvest cells and media with a scrapper.

2) Transfer the cells and media into 50ml tubes.

3) Centrifuge at 500 x g for 5 min to pellet the cells.

4) Discard sup.

5) Add 8.5 ml of Lysis buffer.

6) Cell lysis

- Freeze-thaw cell pellets 3 times at -80°C and 37°C to release the virus into the supernatant.

- Vortex the cell pellets in between the freeze-thaw cycles

7) Add 50U/ml Benzonase and Incubate in 37°C/ 30min

8) Centrifuge at 4000 rpm for 10 min