|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||18916||Plasmid sent as bacteria in agar stab||1||$65|
Virus (100 µL at titer ≥ 1×10¹³ vg/mL)
and Plasmid. More Information
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 5340
Vector typeAAV, Cre/Lox ; Adeno-Associated Virus
Growth in Bacteria
Growth Strain(s)NEB Stable
Growth instructionsStbl2 E coli from Invitrogen Grow at 30 degrees in 2xYT media
Copy numberLow Copy
Gene/Insert nameChannelrhodopsin 2-mCherry
Insert Size (bp)1677
/ Fusion Protein
- mCherry (C terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site Spe1 (destroyed during cloning)
- 3′ cloning site Spe1 (destroyed during cloning)
- 5′ sequencing primer CTGTGGCTGCGTGAAAGCCTTG (Common Sequencing Primers)
Note: We now recommend the AAV-FLEX-ChR2tdtomato-rev construct. It shows less clumping relative to ChR2 labeled with mCherry.
These vectors are prone to recombination. This is a well known issue with these AAV vectors and is due to the inverted terminal repeats (ITRs) required for AAV production. To minimize recombination, we propagate these plasmids in Stbl2 cells from Invitrogen. Also, to minimize recombination, cells should be cultured at 30 C.
Note that these cultures will grow slowly (20 h for minipreps). Better yields and culture times are obtained with 2xYT as the media. This is strongly recommended.
Because recombination may still happen occasionally, we do a panel of restriction digestions to assess whether the ITRs are in tact. Separate digestions with PvuII, Sma1, and SnaB1 should be performed. The expected patterns can be calculated from the attached sequence.
Information for AAV1 (Catalog # 18916-AAV1) ( Back to top )
Ready-to-use AAV1 particles produced from AAV-FLEX-rev-ChR2(H134R)-mCherry (#18916). In addition to the viral particles, you will also receive purified AAV-FLEX-rev-ChR2(H134R)-mCherry plasmid DNA.CAG-driven, cre-dependent, humanized channelrhodopsin H134R mutant fused to mCherry for optogenetic activation. These AAV preparations are suitable purity for injection into animals.
- Volume 100 µL
- Titer ≥ 1×10¹³ 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, AAV1 cap gene
- Buffer PBS + 0.001% Pluronic F-68
- Serotype AAV1
- Purification Iodixanol gradient ultracentrifugation
- Reporter Gene 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 or digital droplet PCR: The number of genome copies in viral preparations was measured by real-time qPCR or by digital droplet PCR. Titering on these preparations was performed by the University of Pennsylvania Vector Core. Read Addgene's AAV Titration by qPCR protocol here.
- Purity of viral preparation: Viral preparations were subjected to polyacrylamide gel electrophoresis (PAGE) followed by SYPRO Red 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.
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:AAV-FLEX-rev-ChR2(H134R)-mCherry was a gift from Scott Sternson (Addgene plasmid # 18916)
For viral preps, please replace (Addgene plasmid # 18916) in the above sentence with: (Addgene viral prep # 18916-AAV1)
For your References section:A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. Atasoy D, Aponte Y, Su HH, Sternson SM. J Neurosci. 2008 Jul 9. 28(28):7025-30. 10.1523/JNEUROSCI.1954-08.2008 PubMed 18614669