DharmaFECT Duo Transfection Reagent

Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Edit-R Cas9 Nuclease protein NLS delivered by DharmaFECT transfection reagents

U2OS-(Ubi)EGFP cells were plated at 10,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 25 nM Edit-R Cas9 Nuclease protein NLS and synthetic crRNA:tracrRNA at 50 or 100 nM targeting PSMD7. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).

Editing of VEGFA gene in HEK293T cells using Edit-R Cas9 Nuclease mRNA delivered by DharmaFECT transfection reagents

HEK293T cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 200 ng of Edit-R-Cas9 mRNA and synthetic crRNA:tracrRNA targeting VEGFA. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. DF1 = DharmaFECT 1, Duo = DharmaFECT Duo, UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).

DharmaFECT Duo is highly effective in co-transfection of plasmid with siRNA or microRNA

DharmaFECT Duo transfection reagent achieves reliable cotransfection results for a variety of experimental goals. The graph demonstrates specific modulation of Renilla luciferase (Rluc) gene:

Rluc expression is similar for Plasmid Alone and Plasmid+Control

Down-regulation of the Rluc as a result of miRNA mimic (plasmid + miRNA mimic)

Excellent knockdown of the Rluc by siRNA (Plasmid + siRNA)

The psiCHECK-2 vector (100 ng/well; Promega) with cloned microRNA recognition sites for miR-28, miR-95, miR-30d, and miR-105, respectively, were complexed alone or with RNAi reagents (10 nM) using DharmaFECT Duo (0.2 μL/well) in MCF-7 cells at 10,000 cells/well (96-well plate). The RNAi reagents were Dharmacon miRIDIAN Mimics (Negative Control, miR-28, miR-95, miR-30d, and miR-105, respectively) or siRNA (Renilla luciferase pool). Firefly and Renilla luciferase expression was assessed at 48 hours using the Dual-Glo Luciferase Assay System (Promega) and normalized to identically treated psiCHECK-2 empty vector.