PurposeCan be used to generate AAV virus that will express tdTomato in the presence of Cre
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||51503||Plasmid sent as bacteria in agar stab||1||$65|
Limited Stock Available, 2 units left
Virus (100µL at titer ≥ 5×10¹² vg/mL)
and Plasmid. More Information
This material is available to academics and nonprofits only.
- Total vector size (bp) 7227
Modifications to backboneA fragment containing the pCAG promoter-FLEX-tdTomato-WPRE was swapped in to replace the CMV promoter-MCS-hGHpA sequence. Total insert size including the two ITRs is 5028 bp.
Growth in Bacteria
Growth Strain(s)NEB Stable
Insert Size (bp)1461
- Promoter pCAG
- Cloning method Restriction Enzyme
- 5′ cloning site Not1 (not destroyed)
- 3′ cloning site Not1 (not destroyed)
- 5′ sequencing primer none
- 3′ sequencing primer none (Common Sequencing Primers)
NOTE: The map generated by Addgene's software is inaccurate. ORF 3 is identified as dTomato, but the entire ORF 2 is actually a codon-optimized version of tdTomato.
Information for AAV Retrograde (Catalog # 51503-AAVrg) ( Back to top )
Ready-to-use AAV Retrograde particles produced from AAV pCAG-FLEX-tdTomato-WPRE (#51503). In addition to the viral particles, you will also receive purified AAV pCAG-FLEX-tdTomato-WPRE plasmid DNA.Cre-dependent (FLEX) tdTomato expression. 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 ≥ 5×10¹² 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
AAV retrograde cap gene
rAAV2-retro helper (plasmid #81070)
- Buffer PBS + 0.001% Pluronic F-68 + 200 mM NaCl
- Serotype AAVrg, encoded by rAAV2-retro helper (plasmid #81070)
- Purification Iodixanol gradient ultracentrifugation
- Reporter Gene tdTomato
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 that only produce amplicons in the FLEX orientation. The PCR products were visualized on an agarose gel for size confirmation.
tdTomato For: GCCACTACCTGGTGGAGTTC
WPRE Rev: GCAGAATCCAGGTGGCAACA
- FLEX orientation
tdTomato For: GCCACTACCTGGTGGAGTTC
WPRE For: CCTTTCCGGGACTTTCGCTTT
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:AAV pCAG-FLEX-tdTomato-WPRE was a gift from Hongkui Zeng (Addgene plasmid # 51503)
For your References section:A mesoscale connectome of the mouse brain. Oh SW, Harris JA, Ng L, Winslow B, Cain N, Mihalas S, Wang Q, Lau C, Kuan L, Henry AM, Mortrud MT, Ouellette B, Nguyen TN, Sorensen SA, Slaughterbeck CR, Wakeman W, Li Y, Feng D, Ho A, Nicholas E, Hirokawa KE, Bohn P, Joines KM, Peng H, Hawrylycz MJ, Phillips JW, Hohmann JG, Wohnoutka P, Gerfen CR, Koch C, Bernard A, Dang C, Jones AR, Zeng H. Nature. 2014 Apr 10;508(7495):207-14. doi: 10.1038/nature13186. Epub 2014 Apr 2. 10.1038/nature13186 PubMed 24695228