曼地亚红豆杉RAPD反应体系与程序的优化

Optimization of the RAPD reaction system and procedure of Taxus media

关键词:  曼地亚红豆杉  RAPD优化  反应体系和程序  正交组合  单因素

Key words:  Taxus media  RAPD optimization  reaction system and procedure  orthogonal design  single factor

摘要:

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:25 μLPCR反应体系中10×Buffer 2.5 μL,MgCl₂ 1.5 mmol/L,dNTP 0.2 mmol/L,随机引物0.6 μmol/L,Taq酶1.0 U,模板DNA 80 ng; 退火温度为37 ℃,循环次数为45次。

Abstract:

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.