Pcdna 3.4 topo ta cloning kit

Full-length human TRAIL cDNA was cloned into the pcDNA3.3-TOPO plasmid using a pcDNA 3.4 TOPO TA Cloning Kit (#A14697; Thermo Fisher Scientific, Inc.) as shown in Fig. 1A. In brief, TRAIL cDNA was amplified from MIGR1-TRAIL-GFP as described by Wiley et al (18 (link)) and then sub-cloned into the pcDNA3.3-TOPO plasmid. The TRAIL cDNA was connected to the pcDNA 3.3-TOPO plasmid with T4 DNA ligase (#D7006; Beyotime Institute of Biotechnology, Beijing, China). After colony polymerase chain reaction (PCR) amplification (the template was bacterium suspension) and DNA sequencing confirmation (performed by Sangon Biotech Co., Ltd., Wuhan, China), this plasmid containing TRAIL cDNA was used for tail PCR and modified with the MEGAscript T7 kit (Ambion; Thermo Fisher Scientific, Inc.). Then, modified TRAIL mRNA was isolated with Ambion Anti-Reverse Cap Analog (ARCA; #AM8045) and purified with Ambion MEGAclear spin columns (Thermo Fisher Scientific, Inc.) and treated with Antarctic Phosphatase (New England Biolabs, Ipswich, MA, USA) to remove residual 5′-triphosphates. The transfection of the TRAIL plasmid into ADSCs was conducted using TransIT-mRNA (Mirus Bio LLC., Madison, WI, USA) according to the manufacturer' instructions and ADSCs transfected with TRAIL-cDNA were defined as TRAIL-ADSCs.