This video is about one of the critical steps in AAV production which is using an iodixanol column gradient
and ultracentrifugation to purify AAV released from a cell culture solution.
We will show you how to load and unload the column, as well as give tips for performing this procedure,
so that you can obtain high-quality purified AAV vector solutions suitable for in vivo injection in your own lab.
The basis of this technique is isopycnic density gradient ultracentrifugation,
which isolates viral particles on the basis of size and density.
A column with 4 densities of iodixonal is prepared by slowly layering the solutions.
The bottom layer will be 60% iodixonal, and the lower density solutions will be subsequently layered on top.
We use a special syringe to layer from the bottom, but you can also add the solutions drop wise and layer from the top.
Press the syringe with a slow and even pressure to load the column, being careful not to mix the solutions.
The rate of loading should be about 1 milliliter every 10 seconds. When you first start a new layer, start slowly
but once you have the layer down you can speed up a bit, because you're no longer dripping directly at the interface
with the risk of mixing the layers. The layers are easy to distinguish because of the color indicators.
Once the column is prepared, you can add your clarified virus containing supernatant to the top.
Again, be careful not to disturb the top layer when loading your supernatant.
Top off the column with PBS and seal it, avoiding air bubbles, which can cause the column to collapse under
the vacuum conditions of the ultracentrifuge. Label the tube a bunch of times in different spots
because the marker easily comes off.
Once the column is sealed, squeeze it gently to confirm the seal is intact.
When loading the rotor check that the lid is adequately greased. Now that the column is prepared,
spin at 350 thousands g for 90 minutes. At these high speeds,
it's necessary to spin in a vacuum to reduce the friction and heat generated.
As the column spins the contents are subjected to centrifugal force. While the individual layers will remain
undisturbed during centrifugation, the macromolecules from your viral supernatant at the top of the column
will migrate through the column. and take up residence within these layers depending on their densities.
The molecules will continue to migrate until their density is equal to the buoyant force of the solution,
which will happen once their density matches that of the surrounding gradient medium.
The different layers of the iodixonal gradient have different concentrations, and thus different densities.
These layers remove contaminants from the clarified supernatant, including proteins and nucleic acid.
Recombinant AAV particles will migrate until they enter the 40% iodixonal layer.
DNA and RNA, which are more dense, will continue migrating until they enter the 60% iodixonal layer.
Average cellular proteins, which are less dense, will stop migrating once they exit the 25% iodixonal layer,
and will appear at the interface as a typical hydrophobic blob, similar to what's seen in phenol chloroform
or other methods of macromolecule purification and extraction.
After the spin is complete, gently remove your column from the ultracentrifuge,
being careful not to disturb the layers.
We know that during the spin the AAV particles from the PBS layer should have migrated down to the 40% iodixonal layer,
so that's where they'll be.
First, we'll puncture the column at bottom of the 40% layer. This is the needle from which the AAV solution will flow.
We like to puncture in the yellow 60% layer, just slightly below the clear 40% layer.
Position the needle far enough into the yellow, 60% layer
Such that none of the AAV containing clear layer gets lost.
But you also don't want to collect too much of the yellow 60% layer,
because this layer has the most iodixonal, which you will hav to remove later in the protocol
Be sure to insert the needle with the bevel pointed up, so that the liquid easily enters the needle from the top.
Position the open end of the needle over your first microfuge tube,
because liquid will start flowing the second we make the second puncture.
Now, we want to make a second puncture, which is what allows air to enter the sealed column and for the solution to flow.
Pierce the top of the column like so. Liquid should immediately start flowing, so be ready to collect.
The flow will be rapid at first, and then slow down.
We've done silver stain analysis on all of our fractions under various conditions.
So we know that the few few fractions we collect from the bottom layer are all going to be clean.
For that reason, we fill these tubes up almost all the way to about 1.1 milliliters.
Once we get close to halfway through, we start spacing the fractions out more, so we can analyze them more carefully.
We collect about 8-10 drops, or 500 microliters per tube. Later you can analyze the fractions by
silver stain, to determine which ones you want to keep, and continue purifying to remove all the iodixonal.
See our protocol page for more details on this step.
Thank you for watching Addgene's AAV purification video. Check out our other protocol pages at
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