Edit-R HDR Plasmid Donor Primers

PCR components of Edit-R Plasmid Donor Kits


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Colony PCR primers for the amplification of the 5′ and 3′ homology arms to confirm proper assembly of the Edit-R HDR Donor plasmid.

Confirmation of proper plasmid assembly is a critical step to ensuring an HDR donor plasmid has been properly assembled and is viable for use as a DNA donor. The Edit-R HDR DNA Donor Plasmid Colony PCR Primer Pairs are essential components of the Edit-R HDR Plasmid Donor Kit and are utilized to confirm that the 5′ and 3′ homology arms are properly inserted in the completed donor template plasmid. Following assembly of DNA donor plasmid from the backbone, insert, and two homology arms; a PCR reaction is carried out across the two homology arms from the backbone to the insert region to ensure proper assembly and orientation.

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-PP-05 Edit-R HDR DNA Donor Plasmid Colony PCR Primer Pair - mKate2    
  Edit-R Colony PCR Primer mKate2 Forward B U-008200-FB-05 5 nmol
  Edit-R Colony PCR Primer mKate2 Reverse A U-008200-RA-05 5 nmol
UK-008000-PP-05 Edit-R HDR DNA Donor Plasmid Colony PCR Primer Pair - Backbone    
  Edit-R Colony PCR Primer Backbone Forward A U-008000-FA-05 5 nmol
  Edit-R Colony PCR Primer Backbone Reverse B U-008000-RB-05 5 nmol

Note: The colony PCR primers 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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Donor plasmid colony PCR

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Colony PCR is utilized to confirm proper insertion of the 5′ (A) and 3′ (B) homology arms following assembly of the HDR donor plasmid for 20 clones. PCR products were run on a 2% agarose gel to confirm proper size and ensure the plasmid(s) selected have both of the required arms.


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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.


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Sequence verification of positive clones

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Following colony PCR, Sanger sequencing was utilized to confirm the sequences of positive clones.