摘要
以构建珍稀物种硬叶兜兰SSR-PCR反应体系,采取正交设计试验对硬叶兜兰SSR体系组分中的Mg2+、基因组DNA、DNA Taq聚合酶、dNTPs、引物5个因素在4个不同程度上进行了组合试验,之后对反应体系中该引物的退火温度在梯度PCR仪上筛选,并进行了最优体系的验证。试验结果显示:体积为10μL的PCR管中最优组合为:模板DNA 20ng,Mg2+3.0mmol/L,dNTPs 0.3mmol/L,引物0.8μmol/L,Taq酶0.8U,10×buffer(Mg2+Free)1μL,引物PR710的退火温度为59.6℃,说明该体系具有较好的实用性。
To construct the SSR--PCR reaction system of the rare species P. micranthum, the five factors （Mg2＋ , template DNA,polymerase,dNTPs and primers）in four levelswere optimized by using the orthogonal design method in P. micranthum SSR reaction system. The annealing temperature of the primer in the reaction system was then screened on a gradient PCR apparatus and the optimal system was verified. The results showed the optimal combina- tion of the PCR reaction system with a total volume of 10μl, 20 ngtemplate DNA , 3.0 mmol/LMg2＋ , 0. 3 mmol/ LdNTPs, 0.8μmol/Lprimer, 0.8 UTaq DNA polymerase and μL10×buffer（Mg2＋ Free）. The Primers PR710 annealing temperature is 59.6℃ ,which proves that the system has good practicability.
出处
《绿色科技》
2017年第21期95-99,共5页
Journal of Green Science and Technology
基金
云南省高校优势特色生态学重点学科建设项目(编号:05000511311)
