dCAS9-VP64_GFP (Plasmid #61422)

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Item	Catalog #	Description	Quantity	Price (USD)
Plasmid	61422	Standard format: Plasmid sent in bacteria as agar stab	1	$75	Add to Cart
Concentrated Lentiviral Prep	61422-LVC	Virus (50µL at titer ≥ 2.5×10⁶ TU/mL) and Plasmid. More Information		$180	Add to Cart

This material is available to academics and nonprofits only.

Backbone

• lenti(AMP)

  (Search Vector Database)

• Mammalian Expression, Lentiviral, CRISPR

• GFP

Growth in Bacteria

• Ampicillin

• 37°C

• Stbl3

• none

• High Copy

Gene/Insert

• dCAS9(D10A,H840A)-VP64_2A_GFP

• Synthetic; S. pyogenes

• D10A and H840A in Cas9

• Promoter
  EF1A

Cloning Information

• Cloning method
  Restriction Enzyme
• 5′ cloning site
  BsiWI
  (not destroyed)
• 3′ cloning site
  EcoRI
  (not destroyed)
• 5′ sequencing primer
  GTT TGG ATC TTG GTT CAT TCT CAA GCC TCA G
• 3′ sequencing primer
  cacatagcgtaaaaggagcaacatag

(Common Sequencing Primers)

Resource Information

• • Not Available to Industry
• • 27 References

Depositor Comments

For additional information, protocols and an activator sgRNA design tool, visit our website: http://sam.genome-engineering.org/

Information for Concentrated Lentiviral Prep (Catalog # 61422-LVC)

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Purpose

Ready-to-use Concentrated Lentiviral Prep particles produced from dCAS9-VP64_GFP (#61422). In addition to the viral particles, you will also receive purified dCAS9-VP64_GFP plasmid DNA.

Delivery

• Volume
  50 µL
• Titer
  ≥2.5×10⁶ TU/mL
• Pricing
  $150 USD for preparation of 50 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
  psPAX2 (plasmid #12260)
• Envelope
  pMD2.G (plasmid #12259)
• Buffer
  PBS
• Selectable Marker
  GFP
• Purification
  Lentivirus was harvested from cell culture medium (DMEM + 10% FBS). Lentiviral particles were then collected by precipitation in polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets containing viral particles were resuspended in PBS.

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

• • Terms of Use for Viral Vectors
• • Not Available to Industry

Viral Quality Control

Titering Method:

• Direct fluorescence: Lenti-X cells were transduced with serial dilutions of 61422-LVC. 96 hours later, GFP-positive cells were counted. Read our fluorescence titering assay protocol here.

Notes:

• Quality control was performed on this lentiviral preparation prior to concentration. Results and explanations of quality control methods can be seen in the Information for Lentiviral Prep section on this page.

Visit our viral production page for more information.

How to cite this plasmid

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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:

  dCAS9-VP64_GFP
  was a gift from

  Feng Zhang

  (Addgene plasmid # 61422 ; http://n2t.net/addgene:61422 ; RRID:Addgene_61422)

  For viral preps, please replace (Addgene plasmid # 61422)
  in the above sentence with:

  (Addgene viral prep # 61422-LVC)

• For your References section:

  Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.
  Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F.
  Nature. 2014 Dec 10. doi: 10.1038/nature14136.
  10.1038/nature14136

  
  PubMed 25494202
  

Measure

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