|- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.
Cells were co-transfected with pCMV-Renilla along with following plasmids: 1.5BV Id1-luciferase (kind gift from Dr. Benezra) and Oct-4 luciferase (described in16). MEF binding site were mutated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent technologies). Following primers were synthesized using a software from https://www.genomics.agilent.com/ for site-directed mutagenesis:
mId1mt-F: 5′-agttcatttctctagaaatttgagtcaggcattccgttcagagtaaaagagagcctgctttgaaatc-3′ mId1mt-R: 5′-gatttcaaagcaggctctcttttactctgaacggaatgcctgactcaaatttctagagaaatgaact-3′.
The luminescence of the lysates was measured using Dual-Luciferase Reporter Assay System (Promega) and read by Veritas microplate luminometer (Turner Biosystems). The Firefly luminescence was normalized to Renilla luminescence. Full paper
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