The Dual-Luciferase Reporter Assay was conducted using the pGL3-promoter vector (Promega, Madison, WI, USA), which contains the Firefly luciferase as the primary reporter and the pRL-TK vector (Promega, Madison, WI, USA), which contains the Renilla luciferase as an internal control. The wild type DNA fragments flanking the significant SNPs (BovineHD2100013266, BovineHD0500004109 and BTB-00042676), which encompassing the putative TF binding sites, were synthesized by Beijing Tsingke Biotech Co., Ltd. under the following rules:5′-GGTACCXXXXXXXXXXXXXCTCGAG-3′. Here, the KpnI restriction site (GGTACC) and the XhoI restriction site (CTCGAG) were introduced at the 5′ end and the 3′ end of the DNA fragments, respectively, for cloning purposes.
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