Blood dna extraction kit

Sample collection has been described in detail elsewhere 8 (link). Genomic DNA was extracted from peripheral blood leukocytes using the Blood DNA extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Briefly, 200 to 300 μl blood samples stored in EDTA tubes at -80°C were equilibrated at room temperature and then mixed with protease K digestion reagent. The reaction mixtures were incubated at 56 °C for 10 minutes, then 100% ethanol was added and the mixture was spun through the extraction column. The column membrane was washed and then the DNA was eluted with 100 μl elution buffer (AE). The isolated DNA concentration was calculated with a Nano-Drop 8000 spectrophotometer (Thermo Scientific). The DNA purity was determined by calculating the A260/A280 and A260/A230 ratios.

Total DNA from frozen peripheral blood was extracted following the phenol-chloroform protocol using a Blood DNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, frozen blood samples were thawed and then mixed with proteinase K, followed by heated at 55°C for 10 min. Next, ethyl alcohol was added into the mixture and vortexed and then centrifuged at 6000 g for 1 min. To isolate the DNA from the mixture, AW2 Buffer from the kit was added and then centrifuged at 20 000 g. The ABI StepOnePlus system (ThermoFisher, Waltham, MA) was utilized for DNA genotyping and data analyses. TaqMan method was used to perform the genotyping. Primers were designed online using the ABI Assay-by-Design service. The amplification conditions set as follows: 2 min at 50°C at the start, 10 min at 95°C, 40 cycles of amplification at 95°C for 15 s, and then 60°C for 60 s. For quality control, duplicates of 20% of the samples were interspersed throughout the plates.