Edit-R mKate2 plasmid donor map with important vector features. The example includes 500 bp homology arms for reference.
Diagram of double-strand break (DSB) repair using the homology-directed repair (HDR) pathway.
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 showing the major process steps and general timing necessary to generate a mKate2 knock-in clonal cell line.
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.
Following colony PCR, Sanger sequencing was utilized to confirm the sequences of positive clones.
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.