曼地亚红豆杉RAPD反应体系与程序的优化

以曼地亚红豆杉为研究对象,采用L16(45)正交组合实验和单因素梯度实验对MgCl2、dNTP、随机引物、Taq酶、模板DNA浓度和退火温度、循环次数等影响RAPD扩增的重要因素进行优化,以期建立最优的RAPD反应体系与程序。实验结果表明,各因素最适条件为:25μLPCR反应体系中10×Buffer2.5μL,MgCl21.5mmol/L,dNTP0.2mmol/L,随机引物0.6μmol/L,Taq酶1.0U,模板DNA80ng;退火温度为37℃,循环次数为45次。

Optimization of the RAPD reaction system and procedure of Taxus media

Regarded Taxus media as the research object,several important factors included MgCl2,dNTP,random primer,Taq polymerase,template DNA and annealing temperature,amplification were optimized by L16(45)orthogonal design experiment and single factor experiment in order to establish optimal RAPD system and procedure.The results showed that a total volume of 25 μL PCR reaction system conditioned 10×Buffer 2.5 μL,MgCl2 1.5 mmol/L,dNTP 0.2 mmol/L,random primer 0.6 μmol/L,Taq polymerase1.0 U,template DNA 80ng,and annealing temperature was 37 ℃,amplification for 45 cycles.