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Protocol: Preparation of Cas9 RNP (ribonucleoprotein), transfection . . . Make a 3 µM working solution of Cas9-NLS by diluting with 1X Cas9 Reaction Buffer or Opti-MEM 3 Form the RNP complexes as follows below: 4 Gently mix the reaction and incubate at room temperature for 10 minutes 2 5 Form the liposome complexes as follows below
Protocol for assessment of the efficiency of CRISPR Cas RNP delivery to . . . In this study we will assess the efficiency and optimize current CRISPR Cas RNP delivery protocols to target cells We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques
CRISPR Editing of Immortalized Cell Lines with RNPs Using Lipofection . . . RNP complexes are formed in Opti-MEM™ I Reduced Serum Medium and can be added directly to cells in culture medium irrespective of antibiotics Following transfection, it is not necessary to remove RNP complexes or add change medium
Opti-MEM™ I Reduced Serum Medium - Thermo Fisher Scientific Opti-MEM™ I Reduced-Serum Medium is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology
Lipofectamine® 2000 Reagent - Thermo Fisher Scientific LipofectamineTM Stem Reagent is a proprietary formulation for transfecting nucleic acids into a wide range of human stem cells LipofectamineTM Stem Reagent should be made in Opti-MEMTM I Reduced Serum Medium and can be added directly to cells in culture growing with or without serum ∤ It is not necessary to remove transfection complexes
CRISPR Cas9 Endonuclease-Mediated Mouse Genome Editing of . . . - Springer Due the superficial tension of Opti-MEM, and voluntary placement of the master mix within the electrode length (to avoid electric field deviation at the edge of the electrodes), we find 9 uL of RNP Opti-MEM to be the ideal volume for electroporation in our electrode system
Transfection of CRISPR Cas9 Nuclease NLS ribonucleoprotein (RNP) into . . . Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine® RNAiMAX NTRODUCTION The CRISPR Cas genome editing system consists of a single guide RNA (sgRNA) and the Cas9 endonuclease To c eate gene disruptions, a single guide RNA is generated to direct the Cas9 nuclease to a specific genomic loc