[rAAV-Lab] Purification of Adeno-associated Virus (AAV) Vectors Using Norgen’s AAV Purification Kit – ID2407 [2019/09/26]

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キット

AAV vectorを精製するキットです。前培養の条件、トランスフェクションの条件、後培養の期間などの条件の記載があります。ハーベトス後のキットによる精製のあと、100kDaのUF膜での濃縮と除菌ろ過で精製サンプルを取得し、AAV vectorからDNA抽出、PCRによる目的遺伝子の確認、動物に投与する条件の記載があります。

Purification of Adeno-associated Virus (AAV) Vectors Using Norgen’s AAV Purification Kit
NORGEN Biotek Corp.

AAV Production

Cell seed 10e7 HEK293 cells/15cm plates, DMEM, 10% FBS, 2mM L-glutamine, penicillin/streptomycin
Transfection (plasmids: rep/cap, genome, helper, containing the inverted terminal repeat (ITR) )after one day
Change media after one day, DMEM+L-glutamine (no serum)
Harvest after 3-6days
Separated Cells and Media

スポンサーリンク: ID6183593457

AAV Purification

For in vitro and vivo, 300mL of AAV9 containing media

33mL fo 300mL ->Norgen AAV purification kit -> eluate (9.9mL) -> concentration 100kDa centrifugal filter unit Amicon Ultra-4 ->0.5mL->0.22μm

スポンサーリンク: ID4219953367

DNA Extraction

5μL + 4μL(2 units) DNase (Norgen’s Kit) -> Incubation 37’c for 30min -> EDTA add final 5mM -> Heating 90’c for 20min (inactivation DNase)

qPCR Quantification

In vitro Transduction with AAV

In vivo administration of AAV

Purified sample -> Concentration (100kDa) -> 4 x 10e11/0.5mL -> 2 divided volume -> 2 mice C57BL/6, ~1 x 10e11/mice -> mice tissues harvesting 1m post transduction ->