*2.2.2. Confirmation of PCR amplification*

Five microliters each of the PCR product obtained, as described above, was subjected to agarose gel electrophoresis, performed using a mini gel electrophoresis system, Mupid (gel: 3% NuSieve 3:1 Agarose; FMC BioProducts or Cambrex Bio Science Rockland). The electro‐ phoresis was carried out at 100 V for 30 min, and the amplification products were visualized under ultraviolet light illumination (Printgraph, Atto Corp.) after staining with ethidium bromide (2 μg/mL) for about 40 min. The presence of the amplified DNA fragment of the intended size was ascertained for each DNA sample. The sizes of DNA fragments amplified using the respective primer sets are described in Table 4.

The purification of the resulting PCR products was performed using the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA, USA) according to the protocol recommended by the manufacturer, as described below. Five volumes of PBI buffer (225 μL) was added to the PCR product (45 μL) and mixed. The resulting solution was placed in the QIA quickspin column and centrifuged at 13,000 rpm (ca. 11,000xg) for 1 min. The supernatant was discarded, 750 μL of PE buffer was added, and the mixture was centrifuged at 13,000 rpm for 1 min. The supernatant was again discarded and the remaining solution was centrifuged at 14,000 rpm (ca. 13,000×g) for 1 min. The supernatant was discarded, the column was transferred to a new 1.5 mL Eppendorf tube, and 30 μL of EB buffer was added. After being left to stand for 1 min, the solution was centrifuged at 13,000 rpm for 2 min to elute the purified PCR product.
