Adenovirus Purification by Ultracentrifugation

Initial Step Gradient

1. In an SW40 polyallomer centrifuge tube, add 2.5 mL of 1.4 g/mL of CsCl.

2. Add 2.5 mL of 1.25 g/mL of CsCl by placing tip of a 5-mL pipette slowly dispensing solution to make two phases.

3. Gently add ~7 mL of cleared vector supernatant on top of 1.25 g/mL CsCl. Leave about 0.5 cm at the top of the tube.

4. Add 0.5 mL of mineral oil on top of cleared vector supernatant.

5. Balance tubes against closest sample and load in rotor. Centrifuge for 90 min at 35,000 rpm, 18°C.

7. Remove tubes from the rotor with forceps.

8. Adenovirus appears as an opaque band at interface of 1.25 g/mL and 1.4 g/mL CsCl. Remove band by piercing the tube about 1 cm below vector with 2-mL syringe loaded with 18-G needle

Second Isopycnic Gradient

1. Add 5–6 mL of 1.34 g/mL of CsCl to the recovered vector band from previous step into a new polyallomer centrifuge tube. Cover with ~0.5 mL of mineral oil.

2. In a second tube add CsCl (1.34 g/mL) to equilibrate tubes. Centrifuge for 18 h at 35,000 rpm, 18°C.

3. Remove tube from rotor and place in black safety tube holder.
Adenovirus r appears as opaque band near the center of the tube. Remove band as above in less than 2 mL. Collect vector by keeping the volume to a minimum.

Desalting Column and Storage

1. Prepare PD-10 column following manufacturer’s instructions. Load up to 2 mL of vector on column.

2. Collect by adding 0.5 mL of PBS 1× Ca++/Mg++. Repeat the step 9–10 times. Label 0.5-mL tubes.

3. The vector is clearly visible as an opaque elute in a final volume of ~2.0–3.5 mL (from aliquots 4 to 7), depending on the initial volume size.

4. Combine the most opaque tubes (excluding the extremities which can be used to isolate DNA for further analysis) and add glycerol to a final concentration of 10%. Another option is to dialyze the vector in several changes of PBS at 4°C.

5. Aliquot (10, 50, and 100 uL) in 0.5-mL tubes and store at −80°C as quickly as possible.