|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||18916||Plasmid sent as bacteria in agar stab||1||$65|
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 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.
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 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