AdPure Adenovirus Purification Kit Protocol
This protocol is optimized for 4 X T75
culture flasks or 2 X 150mm culture dishes (total culture volumne 80ml)
1. Preparation of viral supernatant
1) Plate fresh HEK293 cells using a complete
culture medium
2) When cells reach 90% confluency, change to
a fresh medium (15ml per flask), with which viral stock is diluted
3) When 70~80% cells float (after 3~7 days),
harvest cells by directly pipetting culture medium onto the cells
4) Transfer medium and cells to a tube
5) Centrifuge at 5000xg for 10 minutes at 4℃ and save the
media in a new tube
6) Resuspend cell pellet with 4ml medium
(from Step 5) and divide to four microfuge tubes
7) Break down the cells by repeating three
cycles of freezing/thawing using dry ice and the 37℃ water bath
8) Centrifuge at 10,000xg for 5 minutes and
transfer the supernatant to the media collected (See Step 5)
9) Add 10ul AdNuclease and incubate for 30 minutes at 37℃
10) Clarify using a 0.45um filter (filtration can be hard
unless Step 5 and 8 have been well accomplished)
11) Add 6ml Optimization Buffer to the viral supernatant
(See Step 10) and mix gently
2. Purification Procedure
All purification procedure uses 30ml syringe and
purification filter
1) Rinse the purification filter with 5ml Wash
Buffer
2) Slowly pass the viral supernatant through
the purification filter
3) Wash the filter with 10ml Wash Buffer
4) Wash the filter again with 10ml Wash
Buffer
5) Elute virus with 2ml Elution Buffer
6) [Option] dialyze eluted virus against the
wanted buffer
7) Sterilize using 0.22um sterile syringe
filter
8) Add sterile glycerol to 15% (v/v), mix
well and store at -80℃
(viral titer is
usually 1 X 1011 ~ 1 X 1012 VP/ml)
3. Filter regeneration
The Purification filter can be used ONLY
twice for the same adenovirus
Perform this regeneration step immediately
after elution
1) Wash the filter with 10ml Regeneration
Buffer
2) Wash the filter with 50ml distilled water
3) Wash the filter with 5ml Wash Buffer
4) Wrap the regenerated filter and store 4℃ until next use
