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以苦瓜‘MC9’为试材,采用L16(45)正交实验,对苦瓜SSR-PCR反应体系的Mg2+浓度、dNTPs浓度、模板DNA量、Taq聚合酶量、引物浓度等5个因素的4个水平进行优化试验,并通过正交设计直观分析法对Mg2+浓度、dNTPs浓度、Taq聚合酶量进行了单因素确定。结果表明:最佳反应体系为1μL 10×PCR buffer,Mg2+浓度为1.75mmol·L~(-1),dNTPs浓度为0.25mmol·L~(-1),Taq DNA聚合酶1U,DNA 100ng,引物0.20mmol·L~(-1),ddH2O补足至10μL。
Using the bitter Melon ’MC9’ as experimental materials, four orthogonal experiments were carried out using L16 (45) orthogonal test. The results showed that 4 of 5 factors including Mg2 + concentration, dNTPs concentration, DNA amount, Taq polymerase concentration and primer concentration Level optimization experiments, and by orthogonal design of visual analysis of Mg2 + concentration, dNTPs concentration, the amount of Taq polymerase was determined by a single factor. The results showed that the optimal reaction system was 1μL 10 × PCR buffer, the concentration of Mg2 + was 1.75mmol·L -1, the concentration of dNTPs was 0.25mmol·L -1, the concentration of Taq DNA polymerase 1U, DNA 100ng, 0.20mmol · L ~ (-1), ddH2O make up to 10μL.