Mytaqtmred mix 2x

Genomic DNA from all participants was extracted from whole blood collected on EDTA tubes using GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo-Fisher Scientific, Germany) according to the manufacturer's instructions. The isolated DNA concentration and purity were evaluated by NanoDrop TM 2000 (Thermo-Fisher Scientific, USA). Genotyping of KIR2DS1 and the full-length KIR2DS4 (KIR2DS4-FL) with its variant KIR1D (it is identical to 2DS4 except for a 22 bp deletion in the sequences encoding the second Ig domain; D2) were performed separately by PCR with sequence-specific primers (PCR-SSP) using MyTaq TM Red Mix (2X) (BIOLINE, UK) and primers [21] (link) (Biosearch technologies, UK) on Veriti Dx Thermal Cycler (Thermo-Fisher Scientific, USA) programmed with a 2min denaturation step at 94 °C, followed by 30 cycles of 92 °C for 10 s, 65 °C for 30 s, and 68 °C for 1 min 30 s, followed by 72 °C for 10 min. Annealing temperatures were modified for primers amplifying KIR2DS4 (61 °C) and KIR1D (63 °C). The PCR products along with 100 bp marker were resolved by electrophoresis in 2.5% agarose gel stained with 0.3 μg/ml ethidium bromide. The bands were visualized using UV trans-illuminator at 254 nm (Fig. 1).