Taqman snp genotyping assay 40x

Blood samples were obtained from all subjects, and genomic DNA was isolated with the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The extracted DNA was eluted in TE buffer and stored at - 80 °C for further use. Two SNPs of PD-L1 gene, including rs2297136 (A > G) and rs4143815 (C > G), were examined by the real-time polymerase chain reaction (real-time PCR). The SNPs selection was based on those previously evaluated in relation to cancer or those with evidence of functional significance. The PCR mixture was prepared for each sample by using 10 μL TaqMan™ Universal Master Mix II (Thermo Fisher Scientific, USA), 0.5 μL of TaqMan™ SNP Genotyping Assay (40X) (ready-made assay, Cat. No: 4351379, Thermo Fisher Scientific, USA), 1 μL genomic DNA, and 8.5 μL RNase-free water to reach a total volume of 20 μL. A negative control consisting of 10 μL TaqMan™ Universal Master Mix II, 0.5 μL of TaqMan™ SNP Genotyping Assay (40X), and 9.5 μL RNase-free water was also prepared for each SNP. The real-time PCR cycling was initiated with UNG incubation at 60 °C for 2 min, polymerase activation at 95 °C for 10 min, followed by 40 cycles of (denaturation at 95 °C for 15 s and annealing at 60 °C for 1 min).