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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。
Some steps and extraction components of “two-ball method” for DNA extraction were updated to increase the sensitivity of PCR reaction remarkably. The improvements include: (1) adding the summer spores to a centrifuge tube with two steel balls and a 1.6 g / L Chelex, and the summer spores from the summer sporulation with the dried host mesophyll tissue; (2) Add KOH instead of NaOH, and then vigorously mediate 30 min; (3) the tube through the bottom of a small hole, put a new centrifuge tube, centrifuged briefly thrown the contents. The DNA obtained by this method can be used as a substrate for ITS-nrDNA-PCR, RAPD and SSR. At the same time, the dilution of the supernatant in PCR amplification was analyzed. It was found that RAPD and SSR had good reaction results when the amplification dilution was more than 16 times.