多枝赖草高分子量核DNA的有效制备  

An efficient protocol for extraction of high molecular weight nuclear DNA from Leymus multicaulis

作　　者：周玉雷[1] 徐粤宇[2] 赵茂林[3] 曹致中[1] 王志平[4] 

机构地区：[1]甘肃农业大学草业学院,甘肃兰州730070 [2]首都师范大学生命科学学院,北京100037 [3]北京市农林科学院农业生物技术研究中心,北京100089 [4]北京市土肥工作站,北京100029

出　　处：《草业科学》2007年第10期52-56,共5页Pratacultural Science

基　　金：国家自然科学基金资助项目(30370905和30571135);北京市自然科学基金资助项目(5032009)

摘　　要：以多枝赖草Leymus multicaulis黄化苗幼嫩叶片为材料,在液氮中研磨成粉末,加入冰冷的细胞核抽提缓冲液,经一系列钢制细胞筛过滤,离心分离细胞核,相差显微镜镜检后,用低熔点琼脂糖包埋,蛋白酶K原位裂解,经脉冲场电泳分离,用1%低熔点琼脂糖回收获得了较纯净的分子量大于2 Mb的核DNA,Southern杂交显示没有叶绿体和线粒体DNA的污染。利用该方法制备的高分子量核DNA,可用于后续可转化人工染色体(TAC)文库的构建和基因组分析。The etiolated seedlings of Leymus multicaulis were homogenized by grinding in liquid nitrogen. The nuclei were isolated by suspending the powder in ice-cold nuclei isolation buffer, filtering by a series of steeliness cell griddle on ice and centrifuging before photographed under phase-contrast microscope and embedded in low melting point （LMP） agarose. After digestion with proteinase K in situ and isolation by pulsed-field gel electrophoresis, pure HMW nuclear DNA over 2 Mb was isolated. Southern hybridization indicated there was no protoplast and mitochondria DNA contamination. The HMW nuclear DNA prepared using this method was suitable for transformation artificial chromosome （TAC） library and genome analysis of L. multicaulis

关 键 词：多枝赖草 高分子量核DNA 脉冲场电泳 

分 类 号：S543.9[农业科学—作物学] Q781[生物学—分子生物学]