Editing Adenovirus Purification Clontech (section)

== Purify Adenovirus  ==

<br>1. Equilibrate the Filter Assembly with 5 ml 1X Equilibration Buffer. 

To do this, place the inlet tube into the buffer and draw the buffer into the syringe by pulling back on the plunger. Press lightly on the plunger to push the buffer through the Filter at a flow rate of<br>approximately 3 ml/min (this is approximately 1 drop/sec). 

'''Note: '''Do not force residual air through the Filter. To remove air from the syringe, disconnect the Filter, force the air out of the syringe with the plunger, then reposition the Filter on the syringe. 

2. Place the inlet tube into the clarified lysate, and draw the lysate into the syringe. 

3. Load the adenovirus onto the Purification Filter. This is done by pushing the lysate through the Filter at a rate of ~3 ml/min to allow the virus to bind. 

4. Transfer the inlet tube to a sterile tube containing 20 ml 1X Wash Buffer. Push the Wash Buffer through the filter at a rate of ~3 ml/min 

5. Remove the Filter from the Assembly. 

6. To elute the adenovirus, attach the Filter to a new 5 ml syringe containing 3 ml 1X Elution Buffer. Push 1 ml of Elution Buffer through the filter into a sterile 15 ml conical tube. Incubate the Filter at room temperature for 5 min, then push the remaining elution buffer through. Use residual air in the syringe to push any remaining virus through the Filter. Combine the eluate to obtain approximately 3 ml total. 

7. Determine the adenoviral titer. 

'''Notes:'''<br>â¢ We recommend using the Adeno-X Rapid Titer Kit (Cat. No. 631028).<br>â¢ For information on how to measure the adenoviral particle concentration by its optical density at 260nm (OD260 Assay), please go to www.clontech.com/support/titer 

8. The adenovirus can be used immediately, or aliquoted and stored at â70°C. 

For improved long-term stability, and proper tonicity for in vivo applications, we recommend a buffer exchange of the eluted adenovirus into 1X Formulation Buffer. 

'''1X Formulation Buffer:'''<br>2.5% glycerol (w/v), 25 mM NaCl, and 20 mM Tris-HCl, pH 8.0 <br>(GTS buffer; Hoganson, et al., 2002)<br> 

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