Pegfp n1 plasmid

The gene encoding enhanced GFP was cloned from pEGFP-N1 plasmid (BD Clontech, Palo Alto, CA) and inserted into pET28a expression vector and transferred into BL21 (DE3). When the cells were grown in LB medium at 37°C with the absorbance at 600 nm (OD 600) reached 0.8, a final concentration of 1 mM IPTG was added to induce the EGFP soluble expression at 16°C for another 16 hours, otherwise insoluble form was induced at 37°C for 4 hours. For EGFP soluble expression, the cells were harvested at 5,000 g at 4°C for 20 minutes and resuspended in 30 ml of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ · 12H₂O, 2 mM KH₂PO₄, pH 8.0), and then lysed by sonication on ice (Branson Sonifier 450, USA). The lysate was centrifuged at 12,000 g at 4°C for 20 minutes. The clarified supernatant was collected and purified by Ni-NTA affinity chromatography (GE healthcare) following the instructions. For insoluble expression, cells were harvested at 5,000 g at 4°C for 20 minutes and the pellets were stored at −20°C.

The pEGFP-N1 plasmid expressed enhanced green fluorescent protein, was purchased from BD Biosciences Clontech (Palo Alto, CA, USA). PEI modified magnetic nanoparticles (MNPs) and red fluorescent MNPs were purchased from Chemicell (Berlin, Germany). Porcine kidney 15 (PK15) cell line was purchased from National platform of experimental cell resources for sci-tech (Beijing, China). Agarose gel was purchased from Biowest (Spain). Hind III, DNase|and MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich (Shanghai, China). 24 well plate, 96 well plate, DMEM and fetal bovine serum (FBS) were purchased from Gibco (Germany). Lipofectamine 2000 was purchased from Invitrogen (Germany). Gene Finder was purchased from BioV. Co. Ltd. (Xiamen, China). DAPI staining solution was purchased from Beyotime (Shanghai, China)