Purelink plasmid mini 25 reaction kit

Different upstream regulatory regions of the PRDM16 gene were amplified from U251 DNA using PCR with UltraPF DNA polymerase (GeneCopoeia Inc., Rockville, MD, USA). The PCR fragments were digested with MluI/XhoI and linked to the luciferase-based promoter-less plasmid-pGL3-Enhancer Vector (Promega) to create the following plasmids: pGL3-PRDM16-806/-46, pGL3-PRDM16-506/-46, pGL3-PRDM16-256/-46, pGL3-PRDM16-506/-256 and pGL3-PRDM16-806/-506. The sequences and orientations of the cloned fragments were confirmed by direct DNA sequencing. The plasmids used for transfection were isolated and purified using a Purelink Plasmid Mini 25 Reaction Kit (Invitrogen—Life Technologies, Carlsbad, CA, USA). The promoter activities of these fragments were tested via transient transfection of 1 mg of plasmid DNA into the U251 cell lines using the Lipofectamine 3000. For the luciferase-based assay, the results were normalized against Renilla luciferase activity. At least three independent assays were performed.

Different upstream regulatory regions of the LMO3 gene were amplified from U251 DNA using PCR with UltraPF DNA polymerase (GeneCopoeia Inc., Rockville, MD, USA). The PCR fragments were digested with MluI/XhoI and linked to the luciferase-based promoter-less plasmid-pGL3-Enhancer Vector (Promega) to create the following plasmids: pGL3-LMO3–581/0, pGL3-LMO3–431/0, pGL3-LMO3–281/0, pGL3-LMO3–581/-281, pGL3-LMO3–431/-281, and pGL3-LMO3–581/-431. The sequences and the orientation of the cloned fragments were confirmed by direct DNA sequencing. The plasmids used for transfection were isolated and purified using the Purelink Plasmid Mini 25 Reaction Kit (Invitrogen–Life Technologies, Carlsbad, CA, USA). The promoter activities of these fragments were tested via the transient transfection of 1 mg of plasmid DNA into the U251 cell lines using Lipofectamine2000. For the luciferase-based assay, the results were normalized against the Renilla luciferase activity. At least three-independent assays were performed.