Editing AAV-Production-293T-2 (section)

== PURIFICATION OF AAV2 STOCKS BY HEPARIN AFFINITY COLUMN FOLLOWED BY DESALTING<br>  ==

1. Remove the top cap from the HiTrap heparin affinity column and add a few drops (~200 ÎŒl) of PBS-MK buffer to the top of the column to avoid air bubbles.<br> 

<br>2. Connect the HiTrap Luer adaptor to the top of the column. Some of the buffer will overflow; absorb this with Kimwipes.<br> 

<br>3. Add a few drops (~100 ÎŒl) of PBS-MK buffer to the top Luer adaptor. <br> 

<br>4. Place the 10 ml syringe with 10 ml PBS-MK buffer into the Luer adaptor.<br> 

<br>5. Remove the bottom and snap it off.<br> 

<br>6. Wash the column with PBS-MK buffer. It takes ~8 min to deliver 10 ml PBS-MK.<br> 

<br> CRITICAL STEP Air bubbles must be removed from the liquid in the syringe at all steps before pushing it through the column, as they tend to impede flow. Do not exceed a flow rate of 4 ml/min.<br> 

<br> CRITICAL STEP Each time a step is completed, replace the bottom cap before you remove the syringe from the column, then remove the syringe and add 100 ÎŒl of PBS-MK buffer to the Luer adaptor. When the syringe is placed in the Luer adaptor for the next step,remove the bottom cap.<br> 

<br>7. Load the column using the 10-ml syringe containing the iodixanol fraction (from Step 29 of PROCEDURE). Use a syringe pump and rate of 20 ml/h. Reserve approximately 25â30 ÎŒl before loading on the column for quantitation.<br> 

<br>8. Collect the flow-through in a 14-ml tube.<br> 

<br>9. Wash column with 5 ml of PBS-MK buffer using a syringe pump at the rate of 20 ml/h.<br> 

<br>10. Collect the flow-through in a 14-ml tube.<br> 

<br>11. Elute vector from the column with 2.4 ml of 1 M NaCl/PBS-MK buffer.<br> 

<br>12. Collect the first 0.4 ml in a microcentrifuge tube. This is the âvoid volumeâ.<br> 

<br>13. After this initial 0.4-ml void volume, collect the following fractions: fraction 1 is 1 ml, fraction 2 is 0.5 ml and fraction 3 is 0.5 ml. The vector should come off the column in the first 1.5 ml (fractions 1 and 2).<br> 

<br>14. Desalt after the heparin affinity column purification with a HiTrap desalting column. Wash the HiTrap desalting column with 25 ml of DMEM (or other medium/buffer) in a 30-ml syringe.<br> CRITICAL STEP Do not exceed a flow rate of 5 ml/min.<br> 

<br>15. Prepare the following:<br> A 3-ml syringe containing 1.5 ml of fractions 1 and 2.<br> A 3-ml syringe containing 3 ml of DMEM high glucose (or other medium/buffer to be used when transducing cells).<br> 

<br>16. Position the column to collect fractions into microcentrifuge tubes: the first tube for the 1.5 ml of void flow-through, then âexchangeâ fractions X1âX3 (1 ml each).<br> 

<br>17. Load the 1.5 ml of vector into the 3-ml syringe.<br> 

<br>18. Quickly load the 3 ml of DMEM high glucose or other medium in the second 3-ml syringe.<br> 

<br>19. The vector should be in fractions X1 and X2. Pool fractions X1 and X2 and make 10 aliquots of 200 ÎŒl each (or other aliquot sizes) in microcentrifuge tubes. Prepare 1:10 and 1:100 dilutions of the AAV stock in TE buffer for determining titer later. 

The vector stock and dilutions can be stored at â80 °C indefinitely. If necessary, store stocks in small aliquots and<br>avoid repeated freezing and thawing.<br> 

<br> 

PURIFICATION OF AAV2 STOCKS BY HEPARIN AFFINITY COLUMN FOLLOWED BY DESALTING-2<br> 

<br> 

1. Equilibrate HiTrap heparin affinity column with 10 ml PBS-MK at 1 ml/min. 

2. Load the column with AAV at 0.3 ml/min. 

3. Collect the flow-through in a 14-ml tube. 

4. Wash column with 5 ml of PBS-MK buffer using a syringe pump at the rate of 0.3 ml/min. 

5. Collect the flow-through in a 14-ml tube. 

6. Elute AAV from the column with 2.4 ml of 1 M NaCl/PBS-MK buffer. 

7. Collect the first 0.4 ml in a microcentrifuge tube. This is the âvoid volumeâ. 

8. After this initial 0.4-ml void volume, collect the following fractions: fraction 1 is 1 ml, fraction 2 is 0.5 ml and fraction 3 is 0.5 ml. The AAV should come off the column in the first 1.5 ml (fractions 1 and 2). 

9. Desalt using a HiTrap desalting column. Wash the HiTrap desalting column with 25 ml of PBS in a 30-ml syringe. 

Do not exceed a flow rate of 5 ml/min. 

10. Prepare the following:<br> A 3-ml syringe containing 1.5 ml of fractions 1 and 2.<br> A 3-ml syringe containing 3 ml of PBS. 

11. Position the column to collect fractions into microcentrifuge tubes: the first tube for the 1.5 ml of void flow-through, then âexchangeâ fractions X1âX3 (1 ml each). 

12. Load the 1.5 ml of AAV into the 3-ml syringe. 

13. Quickly load the 3 ml of PBS in the second 3-ml syringe. 

14. The AAV should be in fractions X1 and X2. Pool fractions X1 and X2 and make 10 aliquots of 200 ÎŒl each (or other aliquot sizes) in microcentrifuge tubes. Prepare 1:10 and 1:100 dilutions of the AAV stock in TE buffer for determining titer later. 

The vector stock and dilutions can be stored at â80 °C indefinitely. If necessary, store stocks in small aliquots and avoid repeated freezing and thawing.<br> 

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