The Edit-R HDR Plasmid Donor Kit for insertion of EGFP or mKate2 using the HDR pathway is constructed from a universal plasmid backbone, the DNA insert, and two custom homology arms. Assembly of a custom insert plasmid donor utilizes the same strategy only substituting a user-provided DNA fragment for the insert sequence. These products include the EGFP or 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.
- Fast and efficient assembly of the DNA donor plasmid
- Validated protocols for design, assembly, and confirmation of the donor plasmid
- Utilize the CRISPR Design Tool 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
|Catalog Number||Description||Item Number||Quantity|
|UK-008100-02-10||Edit-R HDR Plasmid Donor & Insert - EGFP|
|Edit-R HDR Plasmid Donor Backbone||U-008300-01-1200||1200 ng|
|Edit-R HDR insert - EGFP||U-008100-01-250||250 ng|
|UK-008200-04-10||Edit-RHDR Plasmid Donor & Insert -mKate2|
|Edit-RHDR Plasmid Donor Backbone||U-008300-01-1200||1200 ng|
|Edit-RHDR Insert - mKate2||U-008200-01-250||250 ng|
|U-008300-01-1200||Edit-R HDR Plasmid Donor Backbone||U-008300-01-1200||1200 ng|
Note: The backbone and EGFP or mKate2 inserts are included with the HDR Plasmid Donor Kit; they may be ordered separately if more material is needed.
LMNA tagged at the N-terminus with EGFP in U2OS cells
U2OS cells were transfected with DharmaFECT Duo, Cas9 mRNA, synthetic crRNA-tracrRNA targeting LMNA, and an EGFP donor plasmid specific to the N-terminus of LMNA containing ~1000 bps of homology per homology arm. Cells were passaged as needed, and imaged 7-days post transfection on an InCell 2200 high content fluorescent microscope.
mKate2-SEC61B fusion in U2OS cells
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.
Materials required for cloning HDR plasmid donors
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.
Dharmacon Edit-R mKate2 knock-in donor
Edit-R mKate2 plasmid donor map with important vector features. The example includes 500 bp homology arms for reference.
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 to acquire mKate2 knock-in clonal cell line
Workflow showing the major process steps and general timing necessary to generate a mKate2 knock-in clonal cell line.