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利用改进的CTAB法提取百里香总DNA,同时采用正交试验法对百里香基因组DNA的RAPD-PCR和ISSR-PCR反应体系及扩增条件进行优化,确定了两种标记技术的最佳PCR体系分别为:20μl RAPD反应体系中,含2.0μL 10×PCR Buffer、1.0mmol/L Mg2+、250μmol/L dNTP、1.5U Taq酶、0.8μmol/L引物和80ng模板DNA。20μl的ISSR-PCR反应体系中,含2μL10×PCR Buffer、2.0mmol/L Mg2+、250μmol/L dNTP、1U Taq酶、1.0μmol/L引物和20ng模板DNA。百里香RAPD-PCR和ISSR-PCR反应体系的建立,为今后百里香属植物的系统分类和遗传多样性分析提供一个标准化程序。
By using improved CTAB method to extract total DNA of thyme, we optimized the RAPD-PCR and ISSR-PCR reaction system and amplification conditions of thyme genomic DNA by orthogonal test. The optimal PCR systems of the two methods were : 20μl RAPD reaction system containing 2.0μl 10 × PCR Buffer, 1.0mmol / L Mg2 +, 250μmol / L dNTP, 1.5U Taq enzyme, 0.8μmol / L primer and 80ng template DNA. 20 μl of ISSR-PCR reaction system contained 2 μl of 10 × PCR Buffer, 2.0 mmol / L Mg 2+, 250 μmol / L dNTP, 1 U Taq enzyme, 1.0 μmol / L primer and 20 ng of template DNA. The establishment of thymus RAPD-PCR and ISSR-PCR reaction system provides a standardized procedure for the future systematic analysis of taxonomy and genetic diversity of Thymus species.