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蚜虫内共生菌基因组DNA提取方法的比较和优化

, PP. 643-648

Keywords: 蚜虫,共生菌,DNA提取

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Abstract:

为探究蚜虫共生菌基因组DNA的提取方案,以桃蚜为试验材料,比较了目前较为常用的4种蚜虫基因组DNA提取方法,从DNA纯度、完整性、PCR扩增效率及稳定性等方面进行了比较和评价,并通过调整蛋白酶K用量和水浴温度及时间对STE法进行了优化。结果表明,4种方法提取的蚜虫共生菌基因组DNA均可用于共生菌的PCR扩增检测;CTAB法和SDS法提取的DNA纯度较高,条带较完整,稳定性相对较高,不易降解,而STE法和PCR缓冲液法操作简便,适于快速提取单头蚜虫共生菌的基因组DNA,但纯度相对较低;可根据试验条件和要求进行选择。STE法优化条件为:用30μLSTE缓冲液将蚜虫匀浆,加入1.5μL20mg/mL蛋白酶K,于56℃水浴1.5h;再加入0.1μL10mg/LRNA酶,于37℃培养1h,95℃下处理5min,5000r/min离心3min,将提取到的DNA于-20℃保存或直接用于PCR扩增。优化后的STE法可作为提取蚜虫共生菌基因组DNA经济而快捷有效的方法。

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