Optimizing SSR-PCR system of Panax ginseng by orthogonal design

Abstract

An orthogonal design was used to optimize SSR-PCR amplification system using Panax ginseng genomic DNA as template. Four levels of five factors (DNA template, Taq DNA polymerase, Mg²⁺, primer, and dNTP) and annealing temperature have been tested separately in this system. The results demonstrated the reaction efficiency was affected by these factors. Based on the results, a stable, productive and reproducible PCR system and cycling program for amplifying a ginseng SSR locus were obtained: 20 µL system containing 1.0 U Taq DNA polymerase, 2.0 mmol·L⁻¹ Mg²⁺, 0.2 mmol·L⁻¹ dNTPs, 0.3 µmol·L⁻¹ SSR primer, 60 ng·µL⁻¹ DNA template, performed with a program of 94°C for 5 min, 94°C for 30 s, annealing at 56.3°C for 30 s, 72°C for 1 min, 37 cycles, finishing at 72°C for 7 min, and storing at 4°C.