Amplicon taq dna polymerase

Ten primers were used for SCoT ampli cation (Table 3). Ampli cation reaction was performed in volumes of 26 µl containing 15.75 µl sterile doubledistilled water, 2.5 µl of the PCR buffer (Amplicon, Cat. No. 180301, 150 mM Tris-HCl pH 8.5, 40 mM (NH4) 2 SO4), 1.5 mM MgCl2, 0.5 mM dNTPs, 0.25 units/ml Amplicon Taq DNA polymerase ('Sigma-Aldrich, USA'), 2.5 pmol of each primer, and 3 µl of template DNA (50 ng/ µl The PCR was carried out at 94°C for 3 min for initial denaturation, 35 cycles of 1 min denaturation at 94°C, 1 min for annealing at 50°C depending on the primer (Table 3), and extension for 1 min 30 second at 72°C. This was followed by a nal extension of 6 min at 72°C. Generated products were separated on 2% agarose gel electrophoresis in 1×TBE buffer and stained with ethidium bromide (10 mg/ml). Fragment size was estimated by using a 1 kb DNA ladder and gels were visualized under UV light.

A set of 34 ISSR primers were used for PCR. Of these primers, only 10 primers were selected based on ampli cation of clear and distinguishable DNA fragments (Table 3). PCR reactions were carried out in a volume of 25 µl, containing 14.75 µl sterile double-distilled water, 2.5 µl of the PCR buffer (Amplicon, Cat. No. 180301), 150 mM Tris-HCl pH 8.5, 40 mM (NH4)2SO4), 1.6 mM MgCl2, 0.5 mM dNTPs, 0.2 units/ml Amplicon Taq DNA polymerase ('Sigma-Aldrich, USA'), 2.5 pmol of primer, and 3 µl of template DNA (50 ng/ µl). The PCR was carried out at 94°C for 3 min for initial denaturation, 45 cycles of 1 min denaturation at 94°C, 1 min for annealing at 50-57°C depending on the primer (Table 3), and extension for 1 min 30 second at 72°C. This was followed by a nal extension of 6 min at 72°C. Generated products were separated on 2% agarose gel electrophoresis in 1×TBE buffer and stained with ethidium bromide (10 mg/ml). Fragment size was estimated by using a 1 kb DNA ladder and gels were visualized under UV light.