AAV-Flex-rev-oChIEF-tdTomato
(Plasmid #30541)

• Purpose: Cre-dependent viral expression of oChIEF-tdTomato for optogenetic excitation
• Depositing Lab: Rory McQuiston
• Publication: Bell et al Neuropharmacology. 2011 Aug 25.

Backbone

• Vector backbone: AAV-FLEX-rev-ChR2-tdtomato
• Backbone manufacturer: Addgene plasmid 18917
• Backbone size w/o insert (bp): 5079
• Vector type: AAV, Cre/Lox

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): Stbl2
• Growth instructions: Stbl2 E coli from Invitrogen Grow at 30 degrees in 2xYT media (see comments section below for more information). Please use 20 ug/ml of Amp.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: oChIEF-tdtomato
• Species: Chlamydomonas reinhardtii
• Insert Size (bp): 1077
• Promoter: CAG
• Tag / Fusion Protein:
  • tdTomato (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: KpnI (not destroyed)
• 3′ cloning site: XbaI (not destroyed)
• 5′ sequencing primer: pCAG-F
• 3′ sequencing primer: WPRE-R

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest
  • Addgene diagnostic digest 2
• A portion of this plasmid was derived from a plasmid made by: Roger Tsien Lab, UCSD for oChIEF-tdtomato
• Articles Citing this Plasmid:
  • 9 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Please note this plasmid has a I170V in channelopsin 1. The changed amino acid improves the protein’s function.

To minimize recombination, propagate this 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.

For more information on growth conditions, please see www.addgene.org/18917 as this is the backbone vector. 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. Please see Reviews in the right column for an image of Addgene's digest with these enzymes.