Edit-R HDR Plasmid Donor Components

Insert and backbone for HDR plasmid construction


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Plasmid backbone and fluorescence insert DNA fragments for construction of an Edit-R HDR donor plasmid

The Edit-R HDR Plasmid Donor Kit for insertion of mKate2 using the HDR pathway is constructed from a universal plasmid backbone, the mKate2 insert, and two custom homology arms. This product includes the mKate2 insert and the plasmid backbone only.

Use and assembly of the donor plasmid requires design of two primer pairs for amplification of the 5′ and 3′ homology arms flanking the insertion site. These primers can be designed and ordered using the HDR Donor Designer.

Highlights

  • Fast and efficient assembly of the DNA donor plasmid
  • Validated protocols for design, assembly, and confirmation of the donor plasmid
  • Utilize the CRISPR Configurator for designing the crRNA to the cut site 
  • Seamless integration with the HDR Donor Designer for designing the primers to create the required 5′ and 3′ homology arms flanking the insertion site

 

Included Components & Amounts

Cat # Description Item # Amount
UK-008200-02-10 Edit-R HDR Plasmid Donor & Insert -mKate2    
  Edit-R HDR Plasmid Donor Backbone - mKate2 U-008200-01-750 750 ng
  Edit-R HDR Insert - mKate2 U-008200-01-250 250 ng

Note: The backbone and insert are included with the HDR Plasmid Donor Kit; they may be ordered separately if more material is needed.

  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C
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Dharmacon™ Edit-R™ mKate2 knock-in donor

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Edit-R mKate2 plasmid donor map with important vector features. The example includes 500 bp homology arms for reference.


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Materials required for cloning HDR plasmid donors

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The Edit-R HDR plasmid donor is created through plasmid cloning and assembly of a fluorescent protein insert sequence, the cut vector backbone, and two homology arms generated with custom PCR primers to geneomc regions flanking the insertion site.


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Diagram of the plasmid donor cloning workflow

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Diagram of the plasmid donor cloning workflow including component ordering, homology arm amplification, and plasmid donor generation. Colors on the diagram indicate the origin of the DNA (dark blue = 5' homology arm, light blue = 3' homology arm, red = mKate2 fluorescent reporter sequence, orange = plasmid backbone). medium alone = media-only negative PCR control.


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Workflow to acquire mKate2 knock-in clonal cell line

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Workflow showing the major process steps and general timing necessary to generate a mKate2 knock-in clonal cell line.


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mKate2-SEC61B fusion in U2OS cells

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Creation of an mKate2 N-terminal fusion to the SEC61B gene, a component of protein transport in the endoplasmic reticulum. U2OS cells were transfected with 50 nM SEC61B-crRNA:tracrRNA, 25 nM Cas9 protein, 2 ng/µL SEC61B mKate2 HDR donor plasmid, and 0.3 µL/well DharmaFECT Duo and visualized on a fluorescent microscope seven days post-transfection. Note: Ribonucleoprotein (RNP) complex ratio is 2:1 for Cas9 protein transfection.