Hi so we have been producing AAV vectors in our lab for some experiments however no one in our lab has been able to concentrate the final virus prep. We have tried both AMICON and VIVASPIN concentrators both with MWCO of 100,000 however we consistently end up with the same titer we started with. Has anyone had success with this? Is our virus getting stuck in the membrane or something? any help would be appreciated.
occasionally we have people in our lab that use our speedvac (vacuum centrifuge) to concentrate vectors. A speedvac generates a vacuum at 45°C -60°C to evaporate liquids. You can ask around if someone in your institute has one standing that you can use. Takes roughly half an hour to go from 500µl to ~50µl
We use Amicon 100KD concentrator post iodixanol and column for AAV vectors. Once the spin is done, we remove the volume containing virus entirely. We the add 100ul of 1xPBS in the empty concentrator and gently wash the both the membrane inside (5 times each side). See if it helps.
Hi Carsten,
we also routinely use Amicon-15, MWCO 100,000 tubes for buffer exchange and simultaneous concentration of iodixanol- and CsCl-purified AAV vectors (all kind of different serotypes and variants), thereby going from ~ 15 mL to ~500 µL. This works perfectly fine and we don't see any significant loss when comparing AAV amounts via qPCR before and after concentration. However, from time to time we try to further concentrate stocks using smaller volume devices. Here we experience much more loss (this might also be in part due to AAV aggregation, which has at least been described for AAV2 at high concentrations and/or low purity). Anyway, I'd be curious to test Tim's suggestion using a speedvac...
Hi there,
I have not had much success with the small volume Amicon (Amicon Ultra-2 or lower), there could be aggregation issues as Benjamin suggested. The larger volume Ultra-4 or Ultra-15 seem to work much better in my experience.
We have also produced a kit for AAV purification and concentration that you could try, which elutes into a final volume of 200 uL. An input of up to 10 mL can be loaded and the kit utilizes mini spin columns for rapid purifications/concentrations using a microcentrifuge. This also makes parallel purification of AAV (20 per mini kit) rapid and easy for screening experiments.
Please send me a message or visit our website for more information:
https://norgenbiotek.com/product/aav-purification-mini-kit
https://norgenbiotek.com/product/aav-purification-mini-kit
@DarrickYu Are these purification kits suitable for purification of AAV8 virus for in vivo experiments?
Hi everyone,
Thanks for the recommendations we have been having much more success using the larger volume spin columns.
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