Nanodrop nd 1000

The 342-bp Igf2 P3 promoter sequence containing the murine Igf2 P3 proximal promoter [33 (link),34 (link)] was synthesized using GeneArt Gene Synthesis (Thermo Fisher Scientific). The transcription start site (TSS) sequence was localized using the Eukaryotic Promoter Database [35 (link)]. The Igf2 P3 promoter sequence was then cloned into the Nanoluciferase expressing promoterless vector pNL1.2[NlucP] (Promega) using NheI (5′) and EcoRV (3′) restriction sites with GeneArt Gene Synthesis. The resulting plasmid was called Igf2 P3 Nluc. Site-directed mutagenesis or promoter deletions were performed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) with 500 nM of each forward and reverse primer (Table 1) and 50 ng of plasmid. PCR was performed at the following conditions: 98 °C for 30 s, followed by 25 cycles at 98 °C for 10 s, for 30 s at 65–71 °C (Table 1), and 72 °C for 150 s and ended at 72 °C for 2 min. The site-directed mutagenesis PCR product was visualized on 1% agarose gel and then treated with the KLD Enzyme Mix (New England Biolabs) prior to transformation into the E. coli strain DH5-α competent cells. Plasmids were extracted with NucleoSpin Plasmid Transfection-grade (Macherey-Nagel), and their concentrations were measured using Nanodrop ND-1000. All plasmids were sequenced using Sanger sequencing (Giga-Genomics).