Mx3005p agilent real time pcr platform

Genotyping of the selected SNVs was performed using the allele discrimination assay on the MX3005p Agilent Real-time PCR platform and we adopted the methodology from our previous studies [4, 6] concentration to 20X using TE (Tris EDTA buffer) as per the manufacturers protocol. The volume of the total PCR reaction was 10 μl, comprising of 2.5 μl of TaqMan UNG Master Mix, 0.25 μl of the probe, 3 μl DNA (5 ng/μl) and 4.25 μl nuclease-free water to make up the nal volume. The following thermal conditions were adopted; hold for 10 min at 95 °C, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All the samples were run in 96-well plates with three no template control (NTC). The post PCR detection system was used to measure allele-speci c uorescence. Ninety-three random samples were picked and re-genotyped for cross-validation of genotyping calls and the concordance rate was 100%.

Genotyping of the selected SNVs was performed using the allele discrimination assay on the MX3005p Agilent Real-time PCR platform and then, methodology was adopted from previous studies [5, 7] . TaqMan Probes rs1002481(VIC: A & FAM: T) rs462779(VIC: A & FAM: G), rs465646(VIC: A & FAM: G), and rs11153292(VIC: A & FAM: G) labelled with VIC and FAM (Thermofisher Scientific) and UNG Master Mix (Applied Biosystems, USA) were used for genotyping. Dilutions of the assay were made from 40X concentration to 20X using TE (Tris EDTA buffer) as per the manufacturers protocol. The volume of the total PCR reaction was 10 μl, comprising of 2.5 μl of TaqMan UNG Master Mix, 0.25 μl of the probe, 3 μl DNA (5 ng/μl) and 4.25 μl nuclease-free water to make up the final volume. The following thermal conditions were adopted; held for 10 min at 95 °C, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All the samples were run in 96-well plates with three no template control (NTC). The post PCR detection system was used to measure allele-specific fluorescence. Ninety-three random samples were picked and re-genotyped for cross-validation of genotyping calls and the concordance rate was 100%.