Qiaquick pcr amplification kit

The genomic region around rs6983267 was amplified via standard PCR on a BioRad T-100 thermal cycler using primers detailed in Fig. S1. The reagents used in each PCR sample included: 3 µl 10X reaction buffer with 1.5 mM MgCl₂, 1 µl 10 mM dNTPs, 1 µl (10 pmole) of each primer, and 0.5 µl Taq polymerase (manufactured by New England BioLabs) with a final reaction volume of 30 µl. The thermal cycler was programmed for five minutes at 95°C for initial denaturation, followed by 35 cycles of 30 sec at 95°C for denaturation, 30 sec at 55°C for annealing, 30 sec at 72°C for extension, and seven minutes at 72°C for the final extension. The PCR amplicon was purified using the QIAGEN QIAquick PCR amplification kit. The purified amplicons were sequenced using conventional Sanger sequencing carried out on an Applied Biosystems Model 3730×l capillary sequencer in the Genome Facility at the University of Iowa Institute of Human Genomics. The results provided the genotype (GG, GT, TT) of each patient for the rs6983267 SNP.