阔叶榧总DNA的提取及其RAPD反应条件的研究

Study on Isolation and Characterization of Total DNA and RAPD Reaction Condition in Torreya fargesii Franch

在阔叶榧新鲜针叶DNA提取过程中 ,应用 pH值较低 ,无机盐浓度较高的提取缓冲液可沉淀蛋白质 ,其中加入 2 %的 β 巯基乙醇可有效地防止次生代谢物质使DNA变色 .提取出的DNA样品产率在 83.7～ 197.5ng/mg .f.,DNA质量和纯度较高 ,2 6 0nm/ 2 80nm光密度比值在 1.8～ 1.9之间 .所得DNA可直接用于限制性内切酶酶切 ,并可用于随机引物PCR扩增 .在优化RAPD各种反应条件研究中 ,确定了阔叶榧最佳RAPD反应体系为 :15 μL反应体系中含有 5 0mmol/LKCl、10mmol/LTris HCl (pH 9.0 )、0 .1%Triton 10 0、3nmol/ μLMgCl2 、0 .6nmol/ μLdNTPs、6 μg/ μLBSA、1UTaq酶、6 0ng引物、5 0～ 10 0ng左右的阔叶榧DNA .

To avoid ionization and oxidation of phenolic compounds during tissue grinding, a low pH extraction buffer with high salts were used to extract total DNA from fresh materials of Torreya fargesii Franch. The yield quality and purity of DNA obtained in new method was enhanad. The extracted DNA could be used directly for RAPD analysis. An optimal reaction system suitable for the assay and usage of RAPD was established. This optimal of 15 μL reaction solution contaioned 50 mmol/L KCl ?10 mmol/L Tris-HCl (pH 9.0)? 0.1% Triton-100?3 nmol/μL MgCl 2?0.6 nmol/μL dNTPs?6 μg/μL BSA?1 U Taq DNA polymerase?60 ng primer and 50～100 ng genomic DNA.