Editing Adenovirus Purification by Q XL

1. Grow 1 × 10<sup>9</sup> 293EF cells&nbsp; in a Nunc cell factory , or 18 of 5 layer (875 cm<sup>2 </sup>&nbsp; ) flasks<br> 

6 well plate==10 cm<sup>2</sup>==&gt;6.25 x 10<sup>5</sup>&nbsp; 293 cells. 1X 10<sup>9</sup>&nbsp;: need 18 of 5-layer multi-flasks (875 cm<sup>2</sup> per flask).<br> 

'''For 1 X 10^12 particles: '''<br> 

3 of 5-layer multi-flasks are required. Total area: 2625&nbsp; cm<sup>2</sup><br> 

Or 15 of T175&nbsp; flasks<br> 

Or 20 of 15 cm dish (135 cm<sup>2</sup>)<br><br> 

Medium: ~500 ml.<br> 

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2. Infect cells with 1 × 10<sup>12</sup> particles of Adenovirus. (1:1000).<br> 

For making 1 X 10<sup>12</sup> particles: <br> 

5 X 10<sup>10</sup> particles of Adenovirus are required<br> 

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3. After 60 hours of infection--&gt; maximum cytopathic effect can be seen.<br> 

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

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

6. Freeze and thaw cells three times.<br> 

7. Spin to remove cell debris.<br> 

8. Load adenovirus in supernantant to a 20 ml Q Sepharose XL column. Flow rate: flow rate: 30 cm/h<br>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<br> 

Identify virus peak by&nbsp; A260/280 ratio.Virus should be eluted by three 5 ml fractions.<br> 

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).<br> 



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

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

11. Concentrate adenovirus using a 100 KDa spin column.<br> 

Yield: <br> 

6.7 x 10^12 particles from 10<sup>9</sup> cells.<br> 

1 X 10^12 particles from 15 of T175 flasks, or 20 of 15 cm dish (135 cm<sup>2</sup>)<br> 

Particle/PFU: 40<br><br><br><br><br><br> 

[[Category:Protocols]]