Pcmv gfp plasmid

The electroporation machine (Catalog# CTX-1500A LE), the pressured electroporation tubes (Catalog# 20 µL: 12–0107; 120 µL: 12–0104; 200 µL: 12–0101), and the electroporation buffer (Catalog# 13–0104) were provided by Celetrix LLC, Manassas VA. Single cell suspensions were prepared as indicated in Cell Culture. Cells were resuspended in electroporation buffer to 25 × 106 cells/ml. For GFP DNA transfection 30 nM pCMV-GFP plasmid (Addgene,Cambridge, MA) was added to the cells. For Cas9 ribonucleoprotein (RNP) transfection, 0.5 μM Cas9-NLS protein (PNA Bio, Newbury Park, CA) was pre-mixed with 0.85 μM gRNA at room temperature for 10 minutes first, then the formed RNP complex was mixed with the cells and transferred to 120 µl electroporation tube. In indicated experiments 2.7 μM ssODN was also added to the mixture. The electroporation conditions were listed in Supplemental Table 1. After electroporation the cells were immediately transferred back to warm medium to continue culture.

Transfection of 2 × 10⁵DUPmyo cells/well of a 6-well plate (10 × 10³ cells/cm²) was performed in serum-free Opti-MEM (Thermo Fisher Scientific) as indicated in manufactures’ instructions. pCMV-GFP plasmid (Addgene #11153) from Connie Cepko’s laboratory was used to test transfection methods. 2.5 μg of plasmid DNA was transfected in combination with different amounts of Lipofectamine 2000 (ThermoFisher Scientific) (6 μl, 9 μl, 12 μl and 15 μl). TurboFect transfection (ThermoFisher Scientific) was done with 2 μg of plasmid DNA mixed with different volumes of transfection reagent (4 μl, 6 μl and 8 μl). 2 μg DNA were also used to transfect cells with 6 μl of GeneJuice reagent (Sigma-Aldrich). After transfection, cells were incubated at 37 °C and 5% CO2 for the amount of time specified by each protocol. Finally, the transfection medium was removed and replaced by the complete Skeletal Muscle Growth medium (PromoCell) and transgene expression evaluated after 48 h.