TITLE: AAV Purification by Iodixanol Gradient Ultracentrifugation
AUTHORS: Addgene the nonprofit plasmid repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol goes through AAV purification by Iodixanol gradient ultracentrifugation. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/aav-purification-iodixanol-gradient-ultracentrifugation/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqim7esn.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqin7esn.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqip7esn.png" /></div></div>

[STEPS]
?. [Procedure]
Preparation and loading of the iodixanol gradient:15% iodixanol step: mix  of 60% iodixanol and  of 1 M NaCl/PBS-MK buffer25% iodixanol step: mix  of 60% iodixanol and  of 1x PBS-MK buffer and 30 μL of phenol red40% iodixanol step: mix  of 60% iodixanol and  of 1x PBS-MK buffer60% iodixanol step: mix  of 60% iodixanol and  of phenol red
4.5 ml
13.5 ml
5 ml
7 ml
6.7 ml
3.3 ml
10 ml
45 µl
?. [Procedure]
Overlay each solution into a QuickSeal tube in the order below using a  syringe and an  needle, taking care to avoid bubbles (Figure 1 sample data in abstract section). of 60% iodixanol step of 40% iodixanol step of 25% iodixanol step of 15% iodixanol step
10 ml
18 g
5 ml
5 ml
6 ml
8 ml
?. [Procedure]
Carefully add up to  of clarified supernatant on top of the gradient. Use 1x PBS (or cell lysis buffer) to top off the tube.
5 ml
?. [Procedure]
Seal the QuickSeal tubes.
?. [Procedure]
Centrifuge at 350,000 g for  in a T70i rotor at .
10 °C
*Pro-Tip*If you need more time, you can alternatively centrifuge for   at 200,000 g at  .
?. [Procedure]
Carefully take the QuickSeal tubes out of the rotor and place them in a stable rack.**Make sure not to disturb the gradient!**
?. [Procedure]
Collect FractionsOption #1Prepare a row of roughly 20 open 1.5 mL microcentrifuge tubes in a rack. These will be used to collect fractions from the gradient. You can collect fewer fractions once you have a good idea when the virus elutes from the gradients.In a biosafety cabinet, carefully puncture the QuickSeal tube at the interface of the 60% and 40% gradient (see Figure 2, in abstract section) with an 18 g needle.Place the first microcentrifuge tube under the needle’s opening to collect the fractions.Puncture the top of the QuickSeal tube with a 16 ga needle and start collecting   to   fractions per tube.Avoid the proteinaceous material near the 40-25% interface. Option #2Puncture the QuickSeal tube at the bottom using an 18 g needle.Immediately start collecting   to   fractions in microcentrifuge tubes.Repeat for each QuickSeal tube.The first   collected corresponds to the 60% phase and can be discarded.The fractions obtained from the 40% phase contain the purified AAV.Option #3Puncture the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a   syringe.The bevel of the needle should be up, facing the 40% step.Collect up to   per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.Repeat for each QuickSeal tube.Puncture the top of the QuickSeal tube with a 16 ga needle and start collecting  to  fractions per tube.Avoid the proteinaceous material near the 40-25% interface. Option #2Puncture the QuickSeal tube at the bottom using an 18 g needle.Immediately start collecting   to   fractions in microcentrifuge tubes.Repeat for each QuickSeal tube.The first   collected corresponds to the 60% phase and can be discarded.The fractions obtained from the 40% phase contain the purified AAV.Option #3Puncture the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a   syringe.The bevel of the needle should be up, facing the 40% step.Collect up to   per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.Repeat for each QuickSeal tube.Option #2Puncture the QuickSeal tube at the bottom using an 18 g needle.Immediately start collecting   to   fractions in microcentrifuge tubes.Repeat for each QuickSeal tube.The first   collected corresponds to the 60% phase and can be discarded.The fractions obtained from the 40% phase contain the purified AAV.Option #3Puncture the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a   syringe.The bevel of the needle should be up, facing the 40% step.Collect up to   per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.Repeat for each QuickSeal tube.Immediately start collecting  to  fractions in microcentrifuge tubes.Repeat for each QuickSeal tube.The first   collected corresponds to the 60% phase and can be discarded.The fractions obtained from the 40% phase contain the purified AAV.Option #3Puncture the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a   syringe.The bevel of the needle should be up, facing the 40% step.Collect up to   per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.Repeat for each QuickSeal tube.The first  collected corresponds to the 60% phase and can be discarded.The fractions obtained from the 40% phase contain the purified AAV.Option #3Puncture the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a   syringe.The bevel of the needle should be up, facing the 40% step.Collect up to   per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.Repeat for each QuickSeal tube.Option #3Puncture the QuickSeal tube slightly below the 60-40% interface with an 18 g needle attached to a  syringe.The bevel of the needle should be up, facing the 40% step.Collect up to   per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.Repeat for each QuickSeal tube.Collect up to  per tube, taking care to avoid collecting the proteinaceous material at the 40-25% interface.
0.5 ml
1 ml
*Pro-Tip*As soon as the top of the tube is punctured, AAV-containing Iodixanol solution will flow from the needle set at the 40-60% interface. Make sure that the microcentrifuge tubes are well positioned to collect the solution or you will lose a significant amount of your virus. When the first fraction is collected, move the rack to the next empty tube to collect the next fraction. The flow will be rapid at first and will progressively slow down.
0.5 ml
1 ml
8 ml
10 ml
5 ml
?. [Procedure]
(Optional) For first time users, it is a good idea to assay each fraction by silver stain to determine purity. Only the cleanest fractions should be kept and pooled for dialysis.
?. [Procedure]
Pool clean fractions.
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Concentration and buffer exchange:Stocks of Pluronic F68:A: 0.1% in PBS:  PBS + Pluronic F68B: 0.01% in PBS:  PBS +  AC: 0.001% in PBS:  PBS +  B + 200 mM NaClProcedure:Cover the filter membrane with of 0.1% Pluronic F68 PBS and incubate for  at room temperatureTake out the 0.1% Pluronic F68 PBS and add of 0.01% Pluronic F68 PBS.Spin at 3000 RPM for  at Discard the flow through and add  of 0.001% Pluronic F68 + 200mM NaCl PBS.Spin at RPM rpm for  at .Spin at 3500 RPM for  at , discard the flow through.Add more sample and spin 3500 rpm for  at , discard the flow through. Repeat this step as needed.9. Use a P1000 to the bottom of the filter and pipette up/down and wash off the walls of the filter to recover as much virus as possible.
49.5 ml
500 µl
45 ml
5 ml
45 ml
5 ml
15 ml
15 ml
4 °C
15 ml
4 °C
4 °C
4 °C
Please note that iodixanol is not easily removed. After each spin, add more formulation buffer and sample and make sure to pipet back and forth a few times to mix the iodixanol that has settled at the bottom of the column.We recommend concentrating to a minimum of 500 µl. If the concentrate volume is less than 500 µl, bring up the volume with formulation buffer.
?. [Procedure]
Store at  for short term (2 weeks), or aliquot and store at  for long term.
4 °C
-80 °C