铁皮石斛野生居群研究(Ⅳ):RAPD反应体系的构建与优化  

Studies of Wild Populations of Dendrobium officinale(Ⅳ): Establishment and Optimization of RAPD Reaction System

作　　者：丁鸽[1] 丁小余[1] 沈洁[1] 刘冬扬[1] 贺佳[1] 唐凤[1] 

机构地区：[1]南京师范大学生命科学学院,江苏南京210097

出　　处：《南京师大学报（自然科学版）》2006年第1期84-88,共5页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30370144)

摘　　要：为获得铁皮石斛RAPD的最佳反应体系,本文对影响RAPD反应的模板含量、Mg2+浓度、dNTP浓度、Taq酶浓度、扩增程序及退火温度等多种因子进行了构建与优化.通过各因子的组合研究,我们得到铁皮石斛RAPD反应最适扩增体系是:25μLPCR反应体积,10×Buffer 2.5μL,2.5 mmol/L MgC l2,dNTP 2.5μL(各2.5mmol/L),模板DNA为30 ng,Taq聚合酶1.5 U,随机引物12 pmol,ddH2O 14.2μL.最佳扩增程序:94℃预变性3m in,94℃变性50 s,38℃复性1 m in,72℃延伸2 m in,循环40次;最后72℃延伸5 m in.4℃保存.To obtain the optimal amplification conditions for Dendrobium officinale, the effects of the concentrations of DNA templates, magesium, dNTP and Taq DNA polymerase and annealing tempreture are tested and optimized. The optimal amplification conditions for Dendrobium officinale is acquired through the experiments： 25 μL PCR reaction volume, 10 × Buffer is 2.5 μL, the concentration of magnesium is 2.5 mmol/L, dNTP is 2.5 mmol/L, the concentration of DNA templates is 30 ng, Taq DNA polymerase is 1.5 U, primer is 12 pmol, ddH20 is 14.2 μL. The optical amplification program is also obtained： 94℃ 180 s, 1 cycle; 94℃ 50 s;38℃ 60 s; 72℃ 120 s, 40 cycles; 72℃ 300 s, 1 cycle, stored in 4℃.

关 键 词：铁皮石斛 随机增扩多态DNA 反应体系 优化 

分 类 号：Q946[生物学—植物学]