Edit-R HDR Plasmid Donor Kits

Kit for assembly of a plasmid DNA mKate2 reporter HDR knock-in donor


Edit-R HDR Plasmid Donor Kits
Everything you need to construct a custom plasmid DNA donor to introduce the mKate2 fluorescent tag to any genomic location using CRISPR-Cas9 and the HDR pathway

Gene engineering using CRISPR-Cas9 relies on two pathways for repair of the cut target DNA, non-homologous end joining (NHEJ) and homology-directed repair (HDR). Precise repair of the DNA to generate a specific mutation or insert or remove a desired sequence requires the HDR pathway. This process is dependent on the availability of a DNA donor template that can be used to repair the double-strand break in the DNA. One of the most common applications is insertion of a fluorescent reporter sequence on one end of a gene. The Edit-R HDR plasmid donor kit enables rapid and easy creation of a plasmid donor for insertion of the mKate2 fluorescence reporter into almost any genomic location.

Highlights

  • Fast and efficient assembly of a 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

The HDR plasmid donor kit contains all the DNA components to build and quality check a custom DNA donor template plasmid to introduce a fluorescent reporter to almost any genomic location when coupled with Edit-R HDR DNA donor plasmid homology arm custom primer pairs.

The kit includes: the Edit-R HDR donor backbone, the Edit-R HDR donor plasmid insert (mKate2), and Edit-R HDR donor plasmid colony PCR primer pairs specific to the mKate2 fluorescent reporter. The HDR Donor Designer is used to design and order the PCR primers for the amplification of the 5′ and 3′ homology arms, required for assembly of the plasmid.

Kit Components & Amounts

Cat # Description Item # Amount
UK-008200-01-10 Edit-R HDR Plasmid Donor Kit -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
  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
  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

 

  
CategoryName
HazardousNo
Hazardous:No
Shelf Life:
Shipping ConditionAmbient
Shipping Condition:
Shipping Information
SpecificationName
SpecificationValue
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C
Storage Condition:
Edit-R-mKate2-HDR-donor-map

Dharmacon™ Edit-R™ mKate2 knock-in donor

Edit-R-mKate2-HDR-donor-map

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


Diagram-of-DSB-repair

Diagram of double-strand break repair

Diagram-of-DSB-repair

Diagram of double-strand break (DSB) repair using the homology-directed repair (HDR) pathway.


Diagram-of-the plasmid-donor-cloning-workflow

Diagram of the plasmid donor cloning workflow

Diagram-of-the plasmid-donor-cloning-workflow

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.


Workflow-mKate2-knock-in-cell-line

Workflow to acquire mKate2 knock-in clonal cell line

Workflow-mKate2-knock-in-cell-line

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


Donor-plasmid-colony-PCR

Donor plasmid colony PCR

Donor-plasmid-colony-PCR

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.


Sequence verification of positive clones

Sequence verification of positive clones

Sequence verification of positive clones

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


mKate2-SEC61B-fusion

mKate2-SEC61B fusion in U2OS cells

mKate2-SEC61B-fusion

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.