Taq dna pol 2x master mix red

The DNA extraction kit manufactured by GeneAll was used to extract and purify the DNA samples. The NanoDrop device (Thermo Scientific, Wilmington, DE, USA) was used to quantify the DNA concentration. The optical density (OD) of the samples was obtained in the absorption rate of 260–280, and it was confirmed if the OD was from 1.8 to 2. Moreover, to check the quality of the extracted DNA, electrophoresis using the agarose gel technique was used. In brief, genomic DNA was amplified by PCR using the Taq DNA Pol 2X Master Mix Red (Cat. no. A180301; Ampliqon, Denmark). The PCR products were sent to GeneAll for DNA sequencing. The quality and average length of the sequence library for each sample were assessed using the Chromas software (version 2.33, http://www.technelysium.com.au/chromas.html).

Genotype determination was carried out by multiplex polymerase chain reaction (multiplex-PCR). Two SNPs spanning the TIM-3 gene were used in our association studies, rs9313439 and rs10515746.
PCR amplification was performed in two separate tubes containing respective reverse primers together with a common forward primer for each allele. The reaction mixture contained 12.5µL of Taq DNA Pol 2x Master Mix Red (Ampliqon; Denmark), 0.5 µL of each forward and reverse primers (10 pmoL/µL), 2µL of template DNA (1 ng/µL), and 8.5 µL autoclaved distilled water, making the final volume 25µL. Polymerase chain reaction was performed using on a programmable thermal cycler (Techne Flexigen - Cambridge, United Kingdom).
The amplification conditions were as follows: a pre-denaturation of 94 ºC for 5 min, 38 cycles of amplification (95 ºC for 1 min, 62 ºC for 1 min, and 72 ºC for 45s) and a final extension reaction was performed at 72 ºC for 10 min. PCR products were separated on 2% agarose gel by electrophoresis. 10% of samples from the study population were randomly selected for direct sequencing analysis to check the accuracy of genotyping.