Bovine CD14 Promoter Amplification

We extracted genomic DNA from 200µL of whole blood utilizing the Isolate II Genomic DNA extraction kit (Bioline USA Inc., Swedesboro, NJ, USA), following the manufacturer’s instructions; a final volume of 100 µL eluted DNA was stored at 4 °C until use and concentration was quantified with a spectrometer (PG Instruments Ltd, England, UK). Specific primer pairs were designed with Primer Express (version 4.0) to amplify the ~1.6 kB promoter region of bovine CD14 gene (Supplementary Table S1). Using primer pairs and EconoTaq Plus Green 2X PCR master mix (Lucigen Corporation, Middleton, WI, USA), we amplified 1 μL of genomic DNA, optimizing reactions to a final volume of 25 μL [16 (link)]. The PCR conditions were programmed as follows: 94 °C for 2 min, and 35 cycles of 94 °C for 30 s, 59 °C for 30 s, 72 °C for 50 s, then the final extension for 5 min at 72 °C. Five microliters of amplified products were examined and band size was determined with a GeneRuler 100bp Plus DNA ladder (Supplementary Table S1). Amplified PCR products (n = 40) were purified with QIAquick PCR purification kit (Qiagen Inc., Valencia, CA, USA), and 2 μL of purified products were prepared for Sanger sequencing (Genewiz, South Plainfield, NJ, USA).