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
出处
《草业科学》
CAS
CSCD
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)
关键词
多枝赖草
高分子量核DNA
脉冲场电泳
Leymus muiticaulis
high molecular weight nuclear DNA
pulsed-field gel electrophoresis
