| 利用单因子和正交设计双重实验方法优化广藿香ISSR-PCR实验体系 |
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| 引用本文： | 曹嵩晓,李娟玲,刘国民,王艺,戴景.利用单因子和正交设计双重实验方法优化广藿香ISSR-PCR实验体系[J].热带生物学报,2011(1):35-41. |
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| 作者姓名： | 曹嵩晓 李娟玲 刘国民 王艺 戴景 |
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| 作者单位： | 海南大学苦丁茶研究所,海南海口570228 |
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| 摘 要： | 为了建立广霍香优化的ISSR-PCR反应体系,首先通过单因子试验选定其各影响因子比较适宜的浓度范围,再利用正交试验设计的方法,对影响广藿香ISSR-PCR反应的5种因素4水平进行优化试验。结果表明：广藿香ISSR-PCR的优化反应体系最终确定为：在25μL反应体系中,含DNA模板40 ng,Mg2＋浓度为2.5 mmol·L^-1,引物浓度为0.3μmol·L^-1,TaqDNA聚合酶用量为1.5 U,dNTPs浓度为150μmol·L^-1。PCR扩增程序为：94℃预变性5 min,然后按94℃变性45 s,52.7℃退火45 s,72℃延伸90 s,进行40个循环,最后72℃延伸7 min,4℃保存。
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| 关 键 词： | 广藿香 ISSR-PCR 单因子试验 正交设计 |
Optimization of ISSR-PCR Experimental System for Pogostemon cablin with the Dual Experiment Methods of the Single Factor and the Orthogonal Design |
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| Institution: | CAO Song-xiao,LI Juan-ling,LIU Guo-min,WANG Yi,DAI Jin(The Kudingcha Research Institute,Hainan University,Haikou 570228,China) |
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| Abstract: | In order to establish the optimized ISSR-PCR experiment system for Pogostemon cablin,the single factor experiments were used to detect the suitable concentration ranges of the different influential factors,and then the orthogonal design was performed to optimize 5 factors（Mg^2＋,dNTPs,primer,Taq polymerase,DNA template） at 4 levels,which affect ISSR-PCR amplification system of Pogostemon cablin.The results indicated that a suitable ISSR-PCR reaction system established： 10× buffer 2.5 μL,40 ng DNA template,2.5 mmo·L^-1MgCl2,0.3 μmo·L^-1primers,1.5 U Taq polymerase,150 μmo·L^-1dNTPs were contained in 25 μL reaction solution.The optimized amplification program was that predenaturing at 94 ℃ for 5 min,then denaturing at 94 ℃ for 45 s,primer annealing at 52.7 ℃ for 45 s,extension at 72 ℃ for 90 s,for 40 cycles,at last extension at 72 ℃ for 7 min,and the products were stored at 4 ℃. |
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| Keywords: | Pogostemon cablin ISSR-PCR single factor tests orthogonal design |
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