GFP, Flag-capsidWT, or Flag-capsidH41R were cloned into AAV vector (Addgene, #37825), and then transfected into HEK293T cells with AAV-PHP.B plasmid (gifted from Dr. Viviana Gradinaru lab) for 3 days to produce AAVs. AAVs-containing supernatants were collected and purified by AAV Purification Mega Kit (Cell Biolabs, #VPK-141). The concentration of purified AAVs was determined by AAV Quantification Kit (Cell Biolabs, #VPK-145) and finally AAVs of 50 μl volume at the concentration of 5×1011 vg/mL was obtained.
Aav purification mega kit
Manufactured by Cell Biolabs
The AAV Purification Mega Kit is a laboratory tool designed for the large-scale purification of adeno-associated virus (AAV) particles. The kit provides the necessary components and protocols to efficiently purify AAV from cell lysates or culture supernatants.
Automatically generated - may contain errors
Lab products found in correlation
Plasmid maxiprep kit, by Qiagen (2 mentions)
Aav vector, by Addgene (1 mentions)
Maxiprep kit, by Qiagen (1 mentions)
Hsyn hm4d gi mcherry, by Addgene (1 mentions)
Paav hsyn dio mcherry, by Addgene (1 mentions)
Paav hsyn dio hm3d gq mcherry, by Addgene (1 mentions)
Paav hsyn dio hm4d gi mcherry, by Addgene (1 mentions)
Paav camkiiα hm4di mcherry, by Addgene (1 mentions)
Paav camkiiα egfp, by Addgene (1 mentions)
4 protocols using aav purification mega kit
1
Production and Purification of AAVs
2
Adeno-Associated Virus Production Protocol
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DNA plasmids encoding pAAV-human synapsin-1 (hSyn)-hM4D(Gi)-mCherry (Addgene, plasmid 50475), pAAV-Ubi-GFP (Addgene, plasmid 11155), and pAAV-Calcium-CaMKIIα-mCherry (Addgene, plasmid 114469) were obtained from Addgene. Plasmid DNA was amplified purified and collected using a standard plasmid maxiprep kit (Qiagen). The purified plasmids were mixed into CaCl2 solution with the DNA plasmid coding AAV-DJ and cotransfected into HEK293T cells using the calcium phosphate precipitation method. Transfected cells were harvested 72 hours after transfection, and the virus was purified using the AAV Purification Mega Kit (Cell Biolabs). Viral titers were 5 × 1012 particles/mL and stored in aliquots at –80°C until use.
3
Production of Viral Vectors Encoding Optogenetic Actuators
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DNA plasmids encoding pAAV-hSyn-DIO-mCherry (Addgene, plasmid #50459), pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, plasmid #44361), pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene, plasmid #44362) and pAAV-mOXT-hM3D(Gq)-mCherry (Addgene, plasmid #70717) were purchased from Addgene. Plasmid DNA was amplified, purified and collected with the QIAGEN plasmid maxiprep kit following manufacturer’s instructions. The purified plasmids were mixed into CaCl2 solution with the DNA plasmid coding AAV5 and co-transfected into HEK293GP cells using the calcium phosphate co-precipitation method as described previously [12 (link), 24 (link)]. Transfected cells were harvested 72 h after transfection and viruses were purified using the AAV Purification Mega Kit (Cell Biolabs, Inc.). Viral titers were 5 × 1012 particles/ml and stored in aliquots at − 80 °C until use.
4
Viral Vector Production and Characterization
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DNA plasmids encoding pAAV-CaMKIIα-hM3Dq-internal ribosomal entry site (IRES)-mCitrine (Addgene, plasmid #50466), pAAV-CaMKIIα-hM4Di-mCherry (Addgene, plasmid #50477), pAAV-CaMKIIα-hChR2(H134R)-EYFP (Addgene, plasmid #26969), pAAV-CaMKIIα-EGFP (Addgene, plasmid #50469), and pAAV-Ubi-eGFP (Addgene, plasmid #62518) were obtained from Addgene. pAAV-CaMKIIα-Cre-GFP was constructed from Cre-GFP empty vector (Addgene, plasmid #20781) into pAAV-CaMKIIα-hChR2-EYFP (Addgene, plasmid #26969). Plasmid DNA was amplified, purified, and collected using a standard plasmid maxiprep kit (Qiagen). The purified plasmids were mixed into CaCl2 solution with the DNA plasmid coding AAV-DJ and co-transfected into HEK293T cells using calcium phosphate precipitation methods. Transfected cells were harvested at 72 h after transfection and the virus was purified using the AAV purification mega kit (Cell Biolabs, Inc.). Viral titers were 5 × 1012 particles/ml and stored in aliquots at −80 °C until use.
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