摘要
为建立高效、快速、准确的烤后烟叶DNA提取方法,采用BioMek NXP自动化工作站与国产DNA磁珠法提取试剂盒相结合,建立了自动化批量提取烤后烟叶基因组DNA（作为PCR模板）的方法,以获取高质量的烤后烟叶DNA满足PCR检测需求。结果表明：水浴时间30min、样本量70-80mg、80%乙醇洗涤4次能获得OD260/OD280为1.7-1.9,浓度大于100ng/μL的烤后烟叶基因组DNA,合格率达95.8%。经PCR定性扩增烟草硝酸还原酶基因（NR）,该方法所提的DNA溶液中无PCR反应抑制物残留,可直接用于PCR定性分析。烟叶粉末经预处理,取上清液上机后约110min可完成96份样品的DNA提取工作。
To establish an efficient,rapid and accurate DNA extraction method from flue-cured tobacco,an automated method for flue-cured tobacco DNA extraction was developed by the BioMek NXP automation workstation combined with magnetic DNA purification kit.Results：Under conditions of water-bath heating 30 min,sample size 70-80 mg and 80% ethanol rinsing four times,OD260/OD280 ratio reached 1.7 - 1.9.Meanwhile,the qualification rate of DNA concentration above 100 ng/ L reached 95.8%.Tobacco NR gene was amplified well and no PCR inhibition was observed,which could be directly used in PCR qualitative analysis.DNA extraction of 96 samples could be finished in 110 min by this method.
出处
《贵州农业科学》
CAS
2015年第5期217-219,222,共4页
Guizhou Agricultural Sciences
基金
贵州省科技支撑计划农业攻关项目"转基因烤烟检测标准物质和检测技术研究及应用"[黔科合NY(2013)3020]
关键词
自动化工作站
烤后烟叶
DNA提取方法
automation workstation
flue-cured tobacco
DNA extraction method
