药用植物草珊瑚RAPD扩增条件优化

Optimization of RAPD amplified conditions in medicinal plant Sarcandra glabra

采用CTAB-DNA提取方法,从草珊瑚植物的嫩叶中提取总DNA。以此DNA为模板,优化了草珊瑚RAPD-PCR的反应条件。结果表明,PCR扩增体系最适宜的条件为:反应体积25μL,内含2.5mmol/L Mg2+、1.0UDNA聚合酶、0.4μmol/L引物、60ng模板DNA和0.16mmol/L dNTP。扩增程序为:94℃预变性2min;94℃变性30s,37℃复性30s,72℃延伸80s,40个循环;72℃延伸10min;4℃保存10min。

The method CTAB-DNA isolation was optimized and used to extract genomic DNA from the tender leaves of Sarcandra glabra. Based on the genomic DNA, some essential factors that affect the result of RAPD were compared and the optimal RAPD system for S. glabra was established. The results showed that the optimal concentration of five important components such as Mg^2＋ , Taq DNA polymerase, primer, template DNA, and dNTP in 25μL RAPD reaction system were 2.5 mmol/L, 1.0U,0. 4 μmol/L, 60 ng,0. 16 mmol/L respectively. Modified thermal profile consisted of an initial denaturation step at 94 ℃ for 2 mins, followed by 40 cycles of 94 ℃ for 30s, 37 ℃ for 30 s,72 ℃ for 80 s,and a final exposure to 72 ℃ for 10mins. Then stored in 4 ℃ for 10 mins.