落叶松-杨栅锈菌DNA提取新方法研究(英文)

A renewed DNA extraction method for molecular study of Melampsora larici-populina

对提取DNA“二球法”的一些步骤和提取成分进行了更新,使得PCR反应灵敏性显著增加。改进的 内容包括:(1)在有二钢珠和1．6 g／L Chelex的离心管中加入夏孢子,夏孢子可以来自带有风干寄主叶肉组织的夏 孢子堆;(2)在离心管中加入KOH而不是NaOH,然后剧烈斡旋30 min;(3)将离心管管底穿一小孔,套上一新离心 管,短暂离心甩出内容物。用该方法得到的DNA可用作ITS-nrDNA-PCR,RAPD和SSR的底物。同时,分析了 PCR扩增中提取上液的稀释倍数,发现RAPD和SSR扩增稀释倍数达到16倍以上有较好的反应结果。

Based on the bi-ball method of DNA extraction,the study renews some steps and component, which makes the PCR more sensible. The changes include.. （1） Put the urediniospores into the tube with two steel balls and 1.6 g/L Chelex-100,the uredinia may be picked up with airing host tissues. （2） Add KOH into the extraction buffer instead of NaOH and vortex the tube violently for 30 min. （3） Prick a hole under the bottom instead of picking out of the steel balls, then centrifuge the tube to toss the liquid into the sheathed tube. The DNA achieved by this way can be used as substrates of ITS-nrDNA-PCR,RAPD,SSR. Futher dilution multiples of the extraction liquid is also tested. RAPD and SSR may complete well with over 16 × DNA extraction buffer.