Super pcr master mix

Genomic DNA was isolated from peripheral blood cells (200 μl whole blood) using a DNA extraction kit (High Pure PCR Template Preparation Kit, Roche, Germany, Cat.No.11796828001) according to manufacturer instructions. The DNA concentration was measured by Nanodrop 2000 (Thermo Fisher, USA). Specific SNPs were genotyped using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) with the PCR reaction performed using super PCR master mix (YEKTA TAJHIZ AZMA, Tehran-Iran, Cat NO: YT1553-YT1554) using a Thermal Cycler instrument (Bio-Rad, CA, USA).
The primer sequences for each PCR reaction is shown in Table 2. The cycle parameters for the PCR analysis were as follows: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. To identify the miR-146 C/G polymorphism, the PCR product was digested with the restriction enzyme mnlI (Thermo Fisher, USA, REF: ER1071) by incubating the samples at 37°C for 4 h. The miR-155 T/A polymorphism PCR product was incubated at 37°C overnight with the restriction enzyme TSP45I (Thermo Fisher, USA, REF: ER1511) and the digestion products were detected by 3% agarose gel electrophoresis.

The boiling method was applied for DNA extraction. A suspension of pure colonies was prepared in 200 µl of distilled water, boiled for 20 min in a water bath, centrifuged for 5 min at 5000 g, and the supernatant preserved at -20 °C until use [26 (link)]. The initial molecular identification was performed by PCR on the ITS1-5.8 S-ITS2 rDNA region via ITS1/ITS4 primer pairs (ITS1: 5′-TCC GTA GGT GAA CCT GCG G-3′; ITS4: 5′-TCC TCC GCT TAT TGA TAT GC-3′) (Sinaclon, Iran). The PCR amplification was done in 25 µl reaction volumes, including 1 µl of each forward and reverse primer, 12.5 µl of super PCR master mix (Yekta Tajhiz Azma, Iran), 3 µl of DNA template, and 7.5 µl of deionized distilled water. Reactions were performed in a thermal cycler PCR system (Peqlab, Germany). The PCR conditions were as follows: an initial cycle of 94 ºC for 5 min, followed by 35 cycles of 94 ºC for 30 s, 56 ºC for 45 s, 72 ºC for 45 s, and a final extension of 72 ºC for 7 min. Finally, the quality of PCR products was evaluated using agarose gel electrophoresis with the Gel Doc XR system (Biorad, USA) and smart ladder (Yekta Tajhiz Azma, Iran). The positive and negative controls were 10 ng/µl of DNA from C. albicans ATCC 10,231 and sterile distilled water, respectively.