FAQ: AAV Purification

Q: Will your AAV Purification kits work with any AAV serotype?

A: Our AAV purification kits use an affinity matrix to purify AAV-2 based on the structure of the AAV2 Cap protein; therefore, they only work for AAV-2 and AAV-DJ and are not suitable for purification of other AAV serotypes.  Other serotypes can be purified by ultracentrifugation.



Q: How do your AAV Purification kits compare to ultracentrifugation

A:  There are three main advantages to using our AAV purification kits: 

1. Our purification kits are easy to use compared to ultracentrifugation, which is very tedious. 

2. Ultracentrifugation is only considered to be partially purified and is used more to concentrate than to purify virus.  Ours kits result in a higher purity compared to ultracentrifugation, which is critical when using AAV in vivo.

3. Our AAV purification kits have a 60% or greater yield; ultracentrifugation has about 40% recovery.



Q: What is the expected titer after using your AAV Purification Kit?

A: Our AAV purification kits are able to recover at least 60% of virus in the supernatant.  The final titer obtained with our purification kits mainly depends on the titer of the crude AAV lysate before the purification.  For these kits to work there must be virus before purification, so optimization of AAV production before purification is very critical.  With optimized transfection conditions, you can get 1-2x1011 GC from one 15cm dish before purification. 



Q: How do your two AAV Purification Kits differ?

A: We offer two AAV Purification Kits: Standard and Mega formats. The difference between these kits is the amount of starting material that can be purified.  The Standard kit was designed to purify supernatant from two 10cm dishes per prep, using a pellet resuspended in 2.5 ml PBS.  The Mega kit (VPK-141), can handle ten 15cm dishes and up to 25ml of viral supernatant per prep.  The amount of viral supernatant you have to be purified should determine which kit is most suitable.



Q: Is the purified AAV suitable for in vivo use?

A: This kit will result in virus that is at least 95% pure, which is a higher purity than ultracentrifugation, and the resulting purified AAV can be used for in vivo studies.



Q: Are the kit reagents sterile?

A: Not all of the solutions provided with our AAV purification kits are sterile.  We recommend filter sterilizing the sample after using the kit if you require a sterile sample.



Q: Do adherent cells need to be harvested? 

A: The adherent cells do need to be harvested and this will require a cell detachment solution.  Our protocol uses EDTA, but trypsin can be used instead. 



Q: Can the Purification Matrix be used more than once?

A: The matrix can only be used once.



Q: How should purified AAV be stored?

A: Adding 5-10% glycerol to purified AAV will increase the stability, but it is not a requirement.  Glycerol should be avoided if your AAV sample will be used in vivo, but if you plan to use it for in vitro applications only it will be fine.  We recommend aliquoting your AAV to avoid freeze/thaw.  The virus will be stable for about 1 year at -80ºC.



Q: How can I improve the results with your AAV Purification Kits?

A: There are two important considerations when using our AAV Purification Kits:

1. Measure the AAV titer before purification. These kits have a virus recovery rate of at least 60%, and the final titer mainly depends on the titer of the crude 293 AAV lysate before starting the purification.

2. The cell pellet must be resuspended in serum-free medium before freeze/thaw cycles to release virus.