欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2009, Vol. 25 ›› Issue (8): 53-57.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

马蓝基因组DNA提取及RAPD反应体系的优化

黄以钟,潘大仁,王占成,陈隽,周以飞   

  • 收稿日期:2009-02-26 修回日期:2009-03-09 出版日期:2009-04-20 发布日期:2009-04-20

Extraction of Genomic DNA and Optimization of RAPD Reaction of Baphicacanthus cusia(Nees)Bremek

, , , , ZHOU Yi-Fei   

  • Received:2009-02-26 Revised:2009-03-09 Online:2009-04-20 Published:2009-04-20

PDF (PC)

858

摘要： 摘 要：以马蓝为研究材料，对马蓝基因组DNA的提取以及RAPD-PCR反应体系进行了优化。结果表明，在提取液中加入PVP和β-巯基乙醇去除酚类物质，采用高盐沉淀DNA和多次洗涤去除糖类，可以得到高质量的马蓝基因组DNA。电泳检测结果表明，所获得的DNA完整，无降解，完全可以满足RAPD等分子标记分析的需要。同时对马蓝RAPD-PCR反应体系中各个影响因素进行了优化，建立了适合马蓝RAPD分子标记的最佳反应体系，即20μl反应体系中含有1.25UTaqDNA聚合酶，0.25mmol/LdNTP，2.5mmol/LMg2+，50ng模板DNA，1.0μmol/L引物；反应程序中最佳退火温度为38℃。

关键词: 硅酸盐细菌, 硅酸盐细菌, 解钾, 溶磷

Abstract: Abstract：The method for extraction genomic DNA from Baphicacanthus cusia(Nees)Bremek and some essential factors affecting the result of RAPD-PCR were investigated in the paper.PVP andβ-mercaptoethanol were added to lyses buffer in order to inhibit oxidation of polyphenol,high concentration of salt solution and elution for many times were used to remove polysaccharide, using above methods ,high quality DNA of Baphicacanthus cusia(Nees)Bremek could be obtained. Based on the high quality genomic DNA,some reaction conditions of PCR for RAPD were optimized.The results showed that the RAPD-PCR system with TaqDNA polymerase 1.25U, dNTP 0.25mmol/L, Mg2+ 2.5mmol/L,genomic DNA 50ng and random primer 1.0μmol/L in 20μl solution was suitable for Baphicacanthus amplification reaction.