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).
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