鳜SSR-PCR反应条件的优化探索

Discussion about the Reaction Conditions of Microsatellite PCR in Siniperca chuatsi

以鳜(Siniperca chuatsi)基因组DNA为试验材料,研究MgCl2浓度、dNTPs浓度、TaqDNA聚合酶3个反应参数对微卫星PCR的影响,建立一套适合鳜微卫星PCR反应的最佳体系。结果表明,在25μL的反应体系中,Mg2+、dNTPs、TaqDNA聚合酶3个参数的适宜浓度分别为1.2mmol/L、0.2μmol/L和0.04U/μL。PCR反应程序为94℃变性5min,94℃30s,56℃1min,72℃1min,共30个循环,最后72℃延伸5min。

In this study, A microsatellite DNA PCR reaction system of Siniperca chuatsi was constructed by the optimization of the concentrations of Mg2＋ ,dNTPS and Taq DNA Polymerase. The results showed that： The optimalized PCR system with total volume 25 μL including MgCl2 at 1.2 mmoL/L, dNTP at 0.2 μmol/L, Taq DNA polymerase at 0.04 U/μL. The PCR reaction program with 5 minutes at 94 ℃ ,which is followed by 30 cycles ,each with 30 seconds at 94℃, 1 minute at 56 ℃, 1 minute at 72℃, and a final extension at 72 ℃ for 5 minutes.