巴东木莲DNA的提取及SRAP-PCR反应体系的正交优化

被引：4

作者：

万玉华 ^{[1
]}

余璐璐 ^{[1
]}

李晓玲 ^{[1
]}

陈发菊 ^{[1
]}

梁宏伟 ^{[1
]}

何正权 ^{[1
]}

机构：

[1] 三峡大学化学与生命科学学院生物技术研究中心/天然产物研究与利用湖北省重点实验室

来源：

湖北农业科学 | 2008年 / 09期

关键词：

巴东木莲; DNA提取; SRAP-PCR反应体系; 正交设计; 优化;

D O I：

10.14088/j.cnki.issn0439-8114.2008.09.002

中图分类号：

Q943.2 [植物基因工程];

学科分类号：

071007 ; 090102 ;

摘要：

研究确定了提取巴东木莲基因组DNA的方法,采用正交试验设计法对影响SRAP-PCR反应的引物浓度、Taq DNA聚合酶的用量、Mg2+和dNTPs浓度及PCR扩增程序中的退火温度及循环次数进行了比较、优化,同时对DNA模板浓度进行了筛选。结果表明,Mg2+、dNTPs、Taq酶及引物的不同水平均对PCR反应结果有显著的影响。建立了巴东木莲20μL SRAP-PCR的反应体系为1×buffer、Mg2+浓度为2.0 mmol·L-1、dNTPs浓度为0.25 mmol·L-1、引物浓度为0.45μmol·L-1、Taq酶为0.5 U和模板DNA 40ng。适宜的扩增程序为94℃预变性1 min,94℃变性1 min,33℃复性1 min,72℃延伸1 min,10个循环;94℃变性1 min,55℃复性1 min,72℃延伸1 min,30个循环;最后72℃延伸5min。试验表明,该体系重复性好、稳定性强。

引用

页码：980 / 984

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