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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (13): 204-207.

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

芹菜RAPD反应体系的优化研究

马建华 杨艳君 郭生金 胡变芳 武青山   

  • 收稿日期:2010-11-30 修回日期:2011-01-05 出版日期:2011-06-05 发布日期:2011-06-05

The Optimization of RAPD Reaction System in Celery

  • Received:2010-11-30 Revised:2011-01-05 Online:2011-06-05 Published:2011-06-05

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摘要：

本试验以一般RAPD反应程序为基础，采用单因素递进筛选方法，针对Taq DNA聚合酶、Mg2+、dNTPs、随机引物和DNA模板5个主要影响因素，分别设置5个不同浓度梯度，对芹菜进行RAPD-PCR扩增，建立了芹菜RAPD技术最优体系。结果表明：25 μL反应体系中含Taq DNA聚合酶1.0 U、Mg2+ 3.0 mM、dNTPs 0.2 mM、引物28 ng、模板DNA 70 ng，10×PCR Buffer 2.5 μL；扩增程序为：94℃预变性5 min，94℃变性1 min，36℃退火1 min，72℃延伸1 min，进行42个循环，最后72℃延伸10 min。

关键词: 原子荧光光谱法, 原子荧光光谱法, 烟叶, 镉

Abstract:

In the present study, we set up separately five different concentration gradient to five main influence factors (dNTPs, Taq DNA polymerase, Mg2+，random primer and DNA template). Then based on the common RAPD reaction system, using the method of single factor progressive screening, we conducted the RAPD-PCR amplification of celery. Finally, we got the optimization of RAPD reaction system in celery. Conclusion of the optimized contents of 25 μL RAPD reaction system was as follows : Taq DNA polymerase 1.0 U, Mg2+ 3.0 mM, dNTPs 0.2 mM，primer 28 ng，DNA templet 70 ng，10×reaction Buffer 2.5 μL. The optimized PCR cycle program was as follows: 94℃ for 5 min, 94℃ for 1 min, 36℃ for 1 min, 72℃ for 1 min, 42 cycles and at final 72℃ for 10 min.