淮山RAPD-PCR反应体系的建立

The Establishment of RAPD-PCR System of Dioscorea opposite Thunb

试验旨在获得最佳的淮山RAPD-PCR反应体系,供试材料来自海南大学农学院淮山资源圃。采用改良CTAB法提取淮山的叶片总DNA,通过单因子实验法分析DNA浓度、引物浓度、dNTP浓度、Taq聚合酶以及Mg2+浓度对RAPD-PCR扩增结果的影响,建立了适合于淮山RAPD-PCR反应体系,即20μL体系中包括:模板DNA为50ng、Taq酶为0.2U、Mg2+浓度为3.0mmol/L、引物浓度为0.2μmol/L、dNTPs浓度为0.2mmol/L。

The purpose of this study was to obtain the best yam RAPD-PCR amplification system,and experimental material resources came from College of Agriculture nursery yam,Hainan University.The total DNAs of Dioscorea opposite Thunb were extracted by the improved CTAB method,and set the gradient by single factor experiment with template DNA concentration,primer concentration,dNTP concentration,Taq concentration and Mg 2 ＋ concentration,Study on the effect of single-factor change on D.opposite RAPD-PCR amplification.In conclusion,the D.opposite reaction system was optimized as below： 50 ng template DNA,0.3 U Taq DNA polymerse,3.0 mmol/L Mg 2＋ ,0.2 μmol/L primer and 0.2 mmol/L dNTPs with total 20 μL reaction solution.