AAV Purification & Concentration Protocol

 

* (D1556-250Ml) OptiPrep™ Density Gradient Medium by Sigma is 60% Iodixanol by W/V

* The following volumes are calculated for the rotor (Beckman-Coulter VTI 70) using 32 ml OptiSeal tubes (Cat # 361625)

 

* 1 M NaCl/PBS-MK buffer

  - Dissolve 5.84 g of NaCl, 26.3 mg of MgCl2and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL.

  - Sterilize by passing through a 0.22-μm filter and store at 4 ℃.

* 1x PBS-MK buffer

  - Dissolve 26.3 mg of MgCl2, and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL.

  - Sterilize by passing through a 0.22-μm filter and store at 4 ℃

 

 

1. Purification

Gradient Purification by Iodixanol

1) Prepare four different gradients (15, 25, 40, 60%)

             - 15% Iodixanol: 4.5 ml of 60% Iodixanol and 13.5 ml of 1 M NaCl/PBS-MK buffer

             - 25% Iodixanol: 5 ml of 60% Iodixanol and 7 ml of 1X-MK buffer and 30 ul of phenol red

             - 40% Iodixanol: 6.7 ml of 60% Iodixanol and 3.3 ml of 1XPBS-MK buffer

             - 60% Iodixanol: 10 ml of 60% Iodixanol and 45 ul of phenol red

2) Overlay each solution into a QuickSeal tube in the order below using a 10ml syringe and an 18g needle, taking care to avoid bubbles.

             a. 6.5 ml of 15% Iodixanol

             b. 6 ml of 25% Iodixanol

             c. 5 ml of 40% Iodixanol

             d. 5 ml of 60% Iodixanol

3) ultra-Centrifuge 80min, 69000rpm (=488727.6 xg), 4’C

4) Collect AAV

- Wipe the outside of the centrifuge tube with 70% ethanol

- Insert an 18G needle ~1 cm from the bottom of the tube at a 90° angle and allow the gradient to drip into sterile microcentrifuge tubes. 

 

 

2. Concentration

1) Raise final volume to 15 ml with 1X DPBS

* Aliquot 1X DPBS before collecting AAV. (9 ml 1X DPBS + 20 μl 10% Pluronic F68)

* Add 5~6 ml of collected AAV sample to the prepared solution.

2) Pre-wet the 100 MWCO filter with 3 ml of 1X PBS and centrifuge 2000 x g for 1 minute

* Can be skipped

3) Load 15ml of column eluate into a filter and centrifuge at 2000 x g for 2 minutes, RT

* If the sample does not flow through, continue to dilute the sample with 1X PBS

4) The concentrated volume should be approximately 250 μl.

* If the concentrated volume is significantly more than 250 μl, discard the flow through and continue to centrifuge in one-minute steps until the volume is approximately right.

5) Add 1X DPBS up to 15 ml and pipette up and down several times

6) Centrifuge until the final volume of the left is 100 μl.

 * Centrifuge 4000rpm, 1hr 30min ~ 2hr (Eppendorf, 5810R)