PurposeAAV-mediated expression of Jaws under the human synapsin promoter
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||65014||Plasmid sent as bacteria in agar stab||1||$65|
Virus (100 µL at titer ≥ 7×10¹² vg/mL)
and Plasmid. More Information
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4695
- Total vector size (bp) 6351
Vector typeMammalian Expression, AAV
Growth in Bacteria
Growth instructionsPlease note that this backbone is highly recombination prone and should be amplified in a rec- strain like Sure2 or Stbl3, and grown at 30 C using carbenicillin rather than ampicillin.
Copy numberLow Copy
SpeciesHaloarcula salinarum (strain Shark)
Insert Size (bp)1650
- Promoter human synapsin
/ Fusion Proteins
- KGC (C terminal on insert)
- GFP (C terminal on insert)
- ER2 (C terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site BamHI (not destroyed)
- 3′ cloning site EcoRI (not destroyed)
- 5′ sequencing primer GCG AGG CGC GAG ATA G
- 3′ sequencing primer CAA GGA GGA GAA AAT GAA AGC C (Common Sequencing Primers)
Information for AAV Retrograde (Catalog # 65014-AAVrg) ( Back to top )
Ready-to-use AAV Retrograde particles produced from pAAV-hsyn-Jaws-KGC-GFP-ER2 (#65014). In addition to the viral particles, you will also receive purified pAAV-hsyn-Jaws-KGC-GFP-ER2 plasmid DNA.These AAV were produced with a retrograde serotype, which permits retrograde access to projection neurons. These AAV preparations are suitable purity for injection into animals.
- Volume 100 µL
- Titer ≥ 7×10¹² vg/mL
- Pricing $350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- Storage Store at -80℃. Thaw just before use and keep on ice.
- Shipment Viral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide
Viral Quality Control
- Real-time qPCR: The number of genome copies in viral preparations was measured by SYBR green real-time qPCR with primers targeting the ITR. Titer values were deduced by comparing the genomic content of the viral preparation to a standard curve of a plasmid of known concentration. Read our AAV Titration by qPCR protocol here.
- Purity of viral preparation: Viral preparations were subjected to polyacrylamide gel electrophoresis (PAGE) followed by silver staining and the molecular weight and relative intensity of the viral capsid proteins was analyzed. The abundance of viral capsid proteins as a fraction of total protein present in the sample was used to determine purity of the AAV preparation.
- PCR confirmation of viral genome: PCR was carried out on the viral preparation with primers targeting the transgene. The PCR products were visualized on an agarose gel for size confirmation.
Syn For: CAAGCACCCAACCCCCATTCCC
37825 Rev: GATATAGACGTTGTGGCTGTTGTAGTTG
Jaws Rev: CTGATCTGGGTGGCCACAAT
37825 Rev: GATATAGACGTTGTGGCTGTTGTAGTTG
Visit our viral production page for more information.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pAAV-hsyn-Jaws-KGC-GFP-ER2 was a gift from Edward Boyden (Addgene plasmid # 65014)
For your References section:Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Chuong AS, Miri ML, Busskamp V, Matthews GA, Acker LC, Sorensen AT, Young A, Klapoetke NC, Henninger MA, Kodandaramaiah SB, Ogawa M, Ramanlal SB, Bandler RC, Allen BD, Forest CR, Chow BY, Han X, Lin Y, Tye KM, Roska B, Cardin JA, Boyden ES. Nat Neurosci. 2014 Aug;17(8):1123-9. doi: 10.1038/nn.3752. Epub 2014 Jul 6. 10.1038/nn.3752 PubMed 24997763