**2.5 Agarose gel electrophoresis**

Agarose gel was prepared by dissolving agarose powder in 1X TBE buffer. The amount of agarose can be dissolved depending upon the purpose in which agarose sheet used. About 0.7% agarose gel was used for visualization of the DNA after extraction while 1.5–2% agarose sheet was used for visualization of PCR product (amplicon). RedSafe (alternative for ethidium bromide) stock solution concentration was 10 mg/ml. Only 5 μl of RedSafe stock solution were added to 100 ml of melted agarose gel to get the final concentration of 0.5 μg/ml [13, 14].

## **2.6 Primer pairs preparation and PCR conditions**

The primers were synthesized at Macrogen/Korea, were provided in a lyophilized form, which were re-dissolved with 300 nuclease-free water according to the institution of the manufacture company to reach to the final concentration (100 pmoles/μl). The working solution will be 10 pmoles/μl to be used directly in PCR [15, 16]. The PCR conditions were calculated using online Protocol Optimize writer software. The conditions were illustrated in **Table 1**.
