Editing Adenovirus Purification Clontech (section)

== Harvest Adenovirus  ==

<br>1. Pellet the cells by centrifugation in a swinging-bucket centrifuge at 1,500 rpm (500 x g for Table-Top Beckman Centrifuge GH3.8 rotor) for 10 min. 

2. Discard the supernatant. 

3. Resuspend the pellet in 5 ml of fresh medium. 

Note: Do not use PBS, as it inhibits some endonucleases. 

Endonuclease is used below to remove contaminating cellular nucleic acids from filter-clarified cell lysate. 

4. Lyse the cells with three consecutive freeze-thaw cycles; briefly freeze the cells in a dry ice/ethanol bath, then thaw them in a 37°C water bath. 

5. After thawing for the final time, centrifuge the lysate at 3,000 rpm (~1,500 x g for Table-Top Beckman Centrifuge GH3.8 rotor) for 5 min to pellet the debris. 

6. Collect and save the supernatant in a sterile centrifuge tube. Discard the pellet. 

7. Add 5 ml Benzonase® Nuclease and incubate for 30 min at 37o C. 

Note: Nuclease treatment decreases the viscosity of the solution so that it can be drawn more easily through the Adeno-X Purification Filter. 

8. Measure the volume of the sample with a pipet, then add an equal volume of 1X Dilution Buffer (approximately 5 ml). 

Note: The solution may turn slightly purple if you resuspended the original cell pellet in fresh medium (step 3). 

9. Clarify the lysate by filtering it through the 0.45 mm Syringe-tip Pre-filter, using a 20 ml syringe (provided).