广东野百合DNA提取和RAPD条件的优化

DNA Extraction and Optimization of RAPD Reaction System for Lilium brownii(Liliaceae)

以野百合(Lilium brownii)新鲜叶片、硅胶干燥叶片及鳞片为材料,研究了DNA的提取方法,并对影响随机扩增多态DNA(RAPD)反应的各因素进行了优化。建立了野百合RAPD的优化反应体系及程序,即在20μl反应体系中,含20 ng模板DNA,2.0 mmol/L Mg2+、0.2 mmol/L dNTPs、1.5 U Taq DNA聚合酶、0.3μmol/L随机引物S1519;扩增程序为:94℃预变性5 min,然后94℃30 s,38℃50 s,72℃1 min,35个循环,最后72℃延伸10 min,4℃保存。

DNA of Lilium brownii was extracted from the fresh leaves, silica gel dried leaves and scales. The optimized reaction system and programme of RAPD for Lilium brownii are as follows. The reactions were performed in a volume of 20 μl containing 20 ng of DNA, 2.0 mmol/L Mg^2＋, 0.2 mmol/L dNTPs, 1.5 U Taq DNA polymerase, 0.3 μmol/L primer （S1519）. Thermal cycling was programmed by 1 cycle for 5 min at 94℃, followed by 35 cycles for 30 sec at 94℃, 50 sec at 38℃, and 1 min at 72℃ and finally, by 1 cycle for 10 min at 72℃, storing at 4℃.