山楂PCR－SSCP反应体系与反应条件的优化
Optimization of PCR-SSCP Reaction System and Reaction Condition in Crataegus spp.
撒云俐 1那冬晨1
作者信息
- 1. 山西师范大学生命科学学院，山西临汾041000
- 折叠
摘要
[目的]优化山楂PCR－SSCP反应体系与反应条件。[方法]以山楂为材料，采用改良的 CTAB 法分步提取叶绿体DNA和核DNA，对PCR－SSCP引物进行筛选，同时进行PCR－SSCP反应体系和反应条件的优化，琼脂糖凝胶电泳检测PCR扩增产物，非变性聚丙烯酰胺凝胶电泳检测变性后的PCR扩增产物。[结果] ropB、ropL、rpoC1、ITS2、psbA－trnH 5对引物适用于山楂PCR－SSCP分析。电泳时，采用6％聚丙烯酰胺凝胶（交联度29∶1），4℃恒温，200 V恒压，上样缓冲液（不含甘油）与PCR产物等量混合，98℃碱（1％NaOH）变性15 min，电泳缓冲液为0．5×TBE，电泳3～4 h可得到较好的山楂PCR－SSCP电泳图谱。[结论]建立了山楂PCR－SSCP最优反应体系与反应条件，为山楂SSCP分析奠定基础。
Abstract
Objective] The aim was to optimize PCR-SSCP reaction system and reaction condition.[ Method] The chloroplast DNA and nu-clear DNA in Crataegus spp.were step-by-step extracted by improved CTAB method, the PCR-SSCP primers were selected, and the PCR-SS-CP reaction system and reaction conditions were optimizated.The PCR-SSCP productions were tested by agarose gelelectrophoresis, the dena-tured PCR-SSCP products were analyzed by native polyacrylamide gel.[ Result] The results showed that five pairs primers ( psbA-trnH、ropB、ropL、rpoC1、ITS2) of all are suitale for PCR-SSCP in Crataegus spp..The clear electrophoretogram of PCR-SSCP in Crataegus spp.were ob-tained through using 6% native polyacrylamide gel.(Crosslinking degree is 29∶1) at 4 ℃and 200 V for 3-4 h, with the volume of the load-ing buffer ( without glycerol) was same with the volume of PCR products, denaturation temperature was 98 ℃ under 1%NaOH, denaturetion time was 15 minutes, electrophoresis buffer was 0.5 ×TBE.[ Conclusion] The study optimize PCR-SSCP reaction system and reaction condi-tion, in order to lay the foundation for optimization of hawthorn SSCP analysis.
关键词
山楂/PCR-SSCP/引物/反应体系/反应条件Key words
Crataegus spp/PCR-SSCP/Primers/Reaction system/Reaction conditions引用本文复制引用
出版年
2016
国家科技期刊平台
NSTL
维普
万方数据