Paul.xu at 23:47, 15 January 2013

2013-01-15T23:47:45Z

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1. Grow 1 × 109 293EF cells  in a Nunc cell factory , or 18 of 5 layer (875 cm2   ) flasks 

6 well plate==10 cm2==>6.25 x 105  293 cells. 1X 109 : need 18 of 5-layer multi-flasks (875 cm2 per flask). 

'''For 1 X 10^12 particles: ''' 

3 of 5-layer multi-flasks are required. Total area: 2625  cm2 

Or 15 of T175  flasks 

Or 20 of 15 cm dish (135 cm2) 

Medium: ~500 ml. 

 

2. Infect cells with 1 × 1012 particles of Adenovirus. (1:1000). 

For making 1 X 1012 particles: 

5 X 1010 particles of Adenovirus are required 

 

3. After 60 hours of infection--> maximum cytopathic effect can be seen. 

4. Collect cells by centrifugation for 20 minutes at 1 000 × g. 

5. Resuspend in 10 or 5 ml of 50 mM Tris-HCl (pH 8.0) in 5% glycerol. 

6. Freeze and thaw cells three times. 

7. Spin to remove cell debris. 

8. Load adenovirus in supernantant to a 20 ml Q Sepharose XL column. Flow rate: flow rate: 30 cm/h 9. Elute with 0 to 1.0 M NaCl gradient in 50 mM Tris/HCl, pH 8.0, in 5% glycerol, for 20 column volumes (CV). 5 ml per fraction. Flow rate: 150 cm/h 

Identify virus peak by  A260/280 ratio.Virus should be eluted by three 5 ml fractions. 

For 16/10 Q XL column, virus should be eluted between 60 ~ 70 min, or 65 min. Fraction is 17, 18 and 19. (vol: 85~95 ml). 

The ratio of 260:280 readings should be close to 1.3 for purified adenovirus. calculate the particle number: OD260 reading x dilution factor x 1.1 x 1012 particles = number of particles per mL of sample. Example: OD260 reading of 0.15 0.15 x 4 x 1.1 x 1012 = 6.6 x 1011 particles per mL of your sample

 

Regenerate the column  with four CV of 0.1 M NaOH between each run. 10. Further purification using a Sartobind IDA membrane unit charged with Zn(2+) ions as affinity ligands. or Gel filtration. 

11. Concentrate adenovirus using a 100 KDa spin column. 

Yield: 

6.7 x 10^12 particles from 109 cells. 

1 X 10^12 particles from 15 of T175 flasks, or 20 of 15 cm dish (135 cm2) 

Particle/PFU: 40 

[[Category:Protocols]]

Adenovirus Purification by Q XL - Revision history

Paul.xu at 23:47, 15 January 2013