马尾松EST-SSR PCR体系的优化

Optimization of PCR system in EST-SSR analysis of Pinus massoniana Lamb

用单因素法研究马尾松EST-SSR PCR体系中各成分及退火温度对扩增结果的影响。结果显示优化后的反应体系为:2μL 10×Buffer(Mg2+Free),30 ng模板DNA,0.187 5 mmol/L-1 dNTPs,3.75 mmol·L-1 Mg2+,8 pmol引物,1.0 U Taq DNA聚合酶,加水至20μL。退火温度为56℃。并用21个马尾松DNA检验这一优化体系。检测试验表明,优化后的EST-SSR PCR反应体系稳定、可靠、可重复性强。

All factors affecting the PCR system in Pinus massoniana Lamb were investigated one by one.The results show that the optimum PCR system of EST-SSR were：2 μL 10×Buffer（Mg2＋ Free）,30 ng template DNA,0.187 5 mmol/L dNTPs,3.75 mmol/ L Mg2＋,8 pmol primer pair,1.0 U Taq DNA poly-merase in total 20 μL reaction system.Finally,a total of 11 isolates of P.massoniana was used for testing the stability of the PCR amplification.The results prove that the optimum PCR system was stable,reliable,highly repetitive.