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adenovirus purification kit and related products.

note: All other components can be stored at 4 ℃ - 25 ℃ Safety Considerations The adenovirus infected cell media and the purified virus can be potential bio-hazardous material and can be infectious to human and animals. All protocols MUST be performed under at least Bio-Safety level 2 working condition.

Materials required but not supplied

• ddH2O
• PBS
• 0.45 μm and 0.22 μm filters
• Rack holder for columns

Adenovirus Purification Kit Protocol

Harvest supernatant from adenovirus-infected cells (For 1-2 T75 flask or equivalent per column)

1. Centrifuge the adenovirus -infected culture media at 3,000 rpm for 10 minutes. Filter the supernatant through a 0.45 μm filter unit.

NOTE: Supernatant from one to two T75 flasks can be processed per column. Up to 1 x1012 virus particles can be purified per column.

2. The supernatant is ready for purification.

NOTE: The supernatant can also be stored at - 80 ℃ for future purification. Equilibrate the column Dilute the 10 x Wash Buffer with dd H2O to 1 x Wash Buffer. Dilute the 2 x Elution Buffer with dd H2O to 1 x Elution Buffer.

3. Set the column in a 15 mL centrifuge tube and spin at 300 x g for 2 minutes. Hold the column with a clamp or other holders. Twist off the bottom and let the liquid drop by gravity flow. Equilibrate the column with 2 mL of dd H2O and then 5 mL 1x Wash Buffer.

NOTE:

• The centrifugation can help remove the bubbles created during shipping.
• A swing-bucket rotor is preferred for centrifugation.
• If the flow-through is too slow, the other alternative is to set the column in a 15 mL conical tube and centrifuge at 400 x g for 1 minute.
• There’s a press-on cap supplied in the kit for the bottom of the column to stop the flow.
• If the flow-through is too slow, make sure to remove any visible bubbles (See trouble shooting on page 6).

Load the adenovirus-containing supernatant to the column

4. Load 5 mL of supernatant to the column and let the supernatant gradually run through the column. Transfer the flow through to another clean tube. Keep loading till all samples pass through the column. Reload the flow through to ensure maximal viral particle binding.

NOTE: If the flow rate gets noticeably slow, cap (the press-on cap to the bottom and the screw cap to the top) and invert the column to mix the supernatant and resin well. Rock the sample for 5 minutes, take off the press-on cap, and put the column into 15 mL tube. Centrifuge at 300 x g for 1 minute. Transfer the flow through to another clean tube if reloading is needed. Keep loading the supernatant till all samples pass through the column. Wash the column and elute the adenovirus

5. Wash the column with 5 mL 1 x Wash Buffer. Repeat once. This step can be performed either by gravity flow or centrifugation at 400 x g.

6. Elute the virus by applying 2-4 mL Elution Buffer. Collect the elution in tubes at 1 mL each. Measure the OD260 and OD280 of each fraction to identify the virus pool. The purified virus should be dialyzed to the desired buffer for downstream application. Sterilize the purified virus by passing through 0.22 μm syringe filter.

7. Aliquot and store the final purified virus at – 80 ℃.

Regeneration of the column

Upon completion of the purification, add 5 mL of Regeneration Buffer to the column by gravity flow and then add 5 mL of 1 x Wash Buffer. Press on the cap to the bottom. Wrap the column with parafilm in a zip block bag and store at 4 ℃.

Adentovirus Purification Troubleshooting Guide

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