锥栗SRAP反应体系的建立与优化

摘要

In order to establish a SRAP-PCR reaction system, and provide theoretical references for the molecular biology research on Castanea henryi in future, taking the genomic DNA of C. henryi leaves as material, the ifve main factors of concentrations of template DNA, Mg2+, dNTP, primer and Taq DNA polymerase in SRAP-PCR ampliifcation system were optimized by using the single factor orthogonal design. The SRAP-PCR ampliifcation conditions suitable for C. henryi are established, containing 40 ng template DNA, 1.8 mmol/L Mg2+, 280μmol/L dNTP, 2.0 U Taq DNA polymerase, 0.6μmol/L primer in 20μL reaction system.%为了建立锥栗SRAP-PCR反应体系，从而为今后的锥栗分子生物学研究提供理论参考，以锥栗叶片基因组DNA为材料，采用单因素试验和正交试验相结合的研究方法，对影响锥栗SRAP-PCR反应体系的模板用量、Mg2＋浓度、dNTP浓度、引物浓度和Taq DNA聚合酶浓度这5个主要因素进行了优化试验。试验确立的适合锥栗的SRAP-PCR反应体系为：20μL体系中，模板DNA用量为40 ng，Mg2＋浓度为1.8 mmol/L，dNTP浓度为280μmol/L，Taq DNA聚合酶浓度为2.0 U，引物浓度为0.6μmol/L。