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pAAV-Ef1a-fDIO-GCaMP6f
(Plasmid #128315)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 128315 Standard format: Plasmid sent in bacteria as agar stab 1 $75
AAV1 128315-AAV1 Virus (100 µL at titer ≥ 1×10¹³ vg/mL) and Plasmid.
$380

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pAAV-Ef1a-fDIO
  • Backbone manufacturer
    Addgene # 55641
  • Backbone size w/o insert (bp) 6323
  • Total vector size (bp) 6965
  • Modifications to backbone
    EYFP cDNA removed by AscI-NheI double digest
  • Vector type
    Mammalian Expression, AAV

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    NEB Stable
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    GCaMP6f
  • Species
    Synthetic
  • Insert Size (bp)
    1371
  • Promoter Ef1a
  • Tag / Fusion Protein
    • His Tag (N terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site AscI (destroyed during cloning)
  • 3′ cloning site NheI (destroyed during cloning)
  • 5′ sequencing primer CACCCACACAAAGGAAAAGGGCC
  • 3′ sequencing primer GCAATAGCATGATACAAAGG
  • (Common Sequencing Primers)

Resource Information

  • Supplemental Documents
  • A portion of this plasmid was derived from a plasmid made by
    GCaMP6f cDNA was obtained as a NotI-BglII fragment from Addgene #40755. Vector backbone was derived from Addgene # 55641.
  • Terms and Licenses
  • Industry Terms
    • Not Available to Industry

Information for AAV1 (Catalog # 128315-AAV1) ( Back to top )

Purpose

Ready-to-use AAV1 particles produced from pAAV-Ef1a-fDIO-GCaMP6f (#128315). In addition to the viral particles, you will also receive purified pAAV-Ef1a-fDIO-GCaMP6f plasmid DNA.

EF1a-driven, Flp-dependent expression of calcium sensor GCaMP6f. These AAV preparations are suitable purity for injection into animals.

Delivery

  • 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

Biosafety

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

Resource Information

Viral Quality Control

Quality Control:
  • Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). The specific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
  • Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.

Visit our viral production page for more information.

Addgene Comments

Using recombinase-dependent vectors in vivo: FRT sites in fDIO plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Flp-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.1-0.8% of viral particles in our typical production protocol. This can lead to a small number of cells exhibiting Flp-independent transgene expression in vivo. To address this, it is necessary to optimize the injection volume and viral titer to find the optimal AAV dosage required for Flp-dependent transgene expression and function in vivo. This may include reducing the viral particle dosage in order to reduce the likelihood of Flp-independent expression.

How to cite this plasmid ( Back to top)

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-fDIO-GCaMP6f was a gift from Patricia Jensen (Addgene plasmid # 128315 ; http://n2t.net/addgene:128315 ; RRID:Addgene_128315)

    For viral preps, please replace (Addgene plasmid # 128315) in the above sentence with: (Addgene viral prep # 128315-AAV1)

  • For your References section:

    A role for the locus coeruleus in the modulation of feeding. Sciolino NR, Mazzone CM, Plummer NW, Evsyukova I, Amin J, Smith KG, McGee CA, Fry SA, Yang CX, Powell JM, Bruchas MR, Kravitz AV, Cushman JD, Krashes MJ, Cui G, Jensen P. bioRxiv 881599v1 10.1101/2019.12.18.881599