仙茅根茎DNA的提取及其ISSR-PCR反应体系的优化

DNA extraction and optimization of ISSR-PCR reaction systems for tubers in Curculigo orchioides

为了改进仙茅基因组DNA的提取方法,从而为其遗传多样性研究提供参考依据,以仙茅根茎为材料,采用CTAB法探讨了仙茅根茎DNA大量提取中CTAB浓度、PVP用量及Na Cl浓度对其DNA质量的影响情况。结果表明,仙茅根茎DNA的大量提取方法的最优组合为:CTAB的浓度为2%,PVP的加入量为0.20 g,Na Cl的浓度为4 mol/L。为了得到仙茅的最佳ISSR-PCR反应体系,以(CT)8 RG为引物,采用单因素实验与正交实验相结合的方法,得到的最优反应体系(25μL)为:1×PCR Buffer,模板DNA浓度为50 ng,Mg2+浓度为2.50 mmol/L,d NTPs浓度为0.20 mmol/L,引物浓度为0.40μmol/L,Taq DNA聚合酶浓度为1.50 U。

In order to improve extraction method of genomic DNA in Curculigo orchioides, and to provide some references for researches on genetic diversity of C. orchioides, taking tubers in C. orchioides as materials, effects of CTAB concentration, PVP dosage and Na Cl concentration on quality of extracted DNA by using CTAB method. The results showed that the optimal DNA extraction combination of tuber in C. orchioides was 2% CTAB, 0.20 g PVP and 4 mol/L Na Cl. To gain the optimal ISSR-PCR reaction system, taking（CT）8RG as primer, some single factor experiments and a orthogonal design experiments were carried out. The optimal reaction system was： 1×PCR Buffer, 50 ng DNA template, 2.50 mmol/L Mg2＋, 0.20 mmol/L d NTPs, 0.40 μmol/L primer, and 1.50 U Taq DNA polymerase with total 25 μL reaction solution.