|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||55634||Plasmid sent as bacteria in agar stab||1||$65|
Virus (100µL 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) 5315
- Total vector size (bp) 7888
Vector typeMammalian Expression, AAV
Growth in Bacteria
Copy numberHigh Copy
Insert Size (bp)708
- Promoter Ef1a
Cloning Information for Gene/Insert 1
- Cloning method Restriction Enzyme
- 5′ cloning site BamHI (not destroyed)
- 3′ cloning site AscI (not destroyed)
- 5′ sequencing primer CACCCACACAAAGGAAAAGGGCC
- 3′ sequencing primer CTTATACACGTGGCTTTTGG (Common Sequencing Primers)
SpeciesM. musculus (mouse)
Insert Size (bp)1272
- Promoter Ef1a/IRES
Cloning Information for Gene/Insert 2
- Cloning method Restriction Enzyme
- 5′ cloning site AscI (not destroyed)
- 3′ cloning site EcoRI (not destroyed)
- 5′ sequencing primer aaccccccacctggcgacag
- 3′ sequencing primer GCAATAGCATGATACAAAGG (Common Sequencing Primers)
Information for AAV Retrograde (Catalog # 55634-AAVrg) ( Back to top )
Ready-to-use AAV Retrograde particles produced from pAAV-EF1a-mCherry-IRES-Flpo (#55634). In addition to the viral particles, you will also receive purified pAAV-EF1a-mCherry-IRES-Flpo plasmid DNA.Flpo and bicistronic (IRES) mCherry expression under the EF1a promoter. 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
- Packaging Plasmids encode adenoviral helper sequences and AAV rep gene
rAAV2-retro helper (plasmid #81070)
- Buffer PBS + 0.001% Pluronic F-68 + 200 mM NaCl
- Serotype AAVrg rAAV2-retro helper (plasmid #81070)
- Purification Iodixanol gradient ultracentrifugation
- Reporter Gene bicistronic mCherry
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.
- Confirmation of protein expression: AAV Pro cells were transduced with 55634-AAVrg. mCherry expression was detected 96 hours later by direct fluorescence.
- 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.
Cherry For: TCCGAGCGGATGTACCCCGAG
FLPo-rev (P415): TGTTCACGATGTCGAAGCTC
- Next-generation sequencing of viral genome: Next-generation sequencing was performed on viral genomes that were isolated from the final viral preparation. Sequencing results were analyzed to confirm the identity and integrity of the viral genome and the absence of unexpected DNA contaminants.
Visit our viral production page for more information.
Addgene CommentsRetrograde functionality is dependent on high viral titers. Addgene recommends not diluting your AAV preps prior to use.
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-EF1a-mCherry-IRES-Flpo was a gift from Karl Deisseroth (Addgene plasmid # 55634)