| 八角RAPD-PCR反应体系的建立及引物筛选研究 |
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| 引用本文： | 陈海云,陈少瑜,宁德鲁,吴涛,耿树香,谷丽萍,白平,李勇杰.八角RAPD-PCR反应体系的建立及引物筛选研究[J].西部林业科学,2012(4):65-69. |
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| 作者姓名： | 陈海云 陈少瑜 宁德鲁 吴涛 耿树香 谷丽萍 白平 李勇杰 |
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| 作者单位： | 1. 云南省林业科学院,云南昆明,650201
2. 云南省林业科学院,云南昆明 650201;国家林业局重点开放性实验室云南珍稀濒特森林植物保护和繁育实验室,云南省森林植物培育与开发利用重点实验室,云南昆明 650201 |
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| 基金项目： | 云南省自然科学基金(2010ZC234) |
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| 摘 要： | 在提取高质量八角基因组DNA的基础上,通过对其RAPD反应体系的Taq DNA聚合酶、dNTPs浓度、Ｍg2+浓度、引物浓度等因子的优化,建立了稳定、重复性高的八角RAPD-PCR反应体系.研究结果表明,在25 μL PCR反应体系中,含1.0 UTaq DNA聚合酶,0.15 mmol·L-1 dNTPs,2.0mmol·L-1 Mg2+,2.0 μmol·L-1引物,20～ 80 ng模板.最佳反应程序为,94℃预变性5 min,扩增后94℃变性30 s,31 ～37℃退火30 s,72℃延伸30 s,35个循环;72℃完全延伸7 min,4℃保存.在建立RAPD-PCR反应体系的基础上,从240条RAPD引物中筛选出15条扩增条带清晰、稳定性好的引物,并通过各引物的退火温度梯度实验,确定了各引物的最适退火温度.
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| 关 键 词： | 八角 RAPD-PCR 反应体系 优化 引物 |
Establishment of RAPD-PCR Reaction System and Primer Selection |
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| CHEN Hai-yun
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CHEN Shao-yu
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NING De-lu
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WU Tao
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GENG Shu-xiang
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GU Li-ping
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BAI Ping
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LI Yong-jie.Establishment of RAPD-PCR Reaction System and Primer Selection[J].Journal of West China Forestry Science,2012(4):65-69. |
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| Authors: | CHEN Hai-yun CHEN Shao-yu NING De-lu WU Tao GENG Shu-xiang GU Li-ping BAI Ping LI Yong-jie |
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| Institution: | 1(1.Yunnan Academy of Forestry,Kunming Yunnan 650201,P.R.China;2.Yunnan Laboratory for Conservation of Rare,Endangered & Endemic Forest Plants,Public Key Laboratory of the State Forestry Administration;Yunnan Provincial Key Laboratory of Cultivation and Exploitation of Forest Plants,Kunming Yunnan 650201,P.R.China) |
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| Abstract: | The RAPD-PCR reaction system of Illicium verum was established through optimizing a series of factors including Taq,the concentration of dNTPs,the concentration of Mg2+ and the concentration of primers.The results indicated that the optimized reaction system of 25 μL was 1.0 U Taq polymerase,0.15 mmol·L-1 dNTPs,2.0 μmol·L-1 primer,20~80 ng template DNA.The proper PCR reaction conditions were as follows: predenaturing at 94 ℃ for 5 min,followed by 35 cycles of denaturing at 94 ℃ for 30 s,annealing at 37 ℃ for 30 s,extension at 72 ℃ for 30 s,and final extension at 72 ℃ for 7 min.Using the established reaction system,fifteen stable primers with clear band were screened out from 240 tested primers,and the optimum annealing temperatures for different primers were developed. |
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| Keywords: | Illicium verum RAPD-PCR reaction system optimization primer |
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