| 甜槠ISSR-PCR 反应体系的正交优化 |
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| 引用本文： | 张文标,金则新,李钧敏,潘冠琼. 甜槠ISSR-PCR 反应体系的正交优化[J]. 浙江农林大学学报, 2006, 23(5): 516-520 |
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| 作者姓名： | 张文标 金则新 李钧敏 潘冠琼 |
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| 作者单位： | 1.西南师范大学三峡库区生态环境教育部重点实验室, 重庆400715;2.台州学院生态研究所, 浙江临海317000 |
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| 基金项目： | 浙江省自然科学基金资助项目(Y504220) |
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| 摘 要： | 对甜槠Castanopsis eyrei 进行遗传多样性研究的过程中, 为了获得清晰可靠、重复性强的ISSR 扩增结果, 利用正交设计对Mg 2+ , dNTP , 引物, Taq 酶, 牛血清蛋白(BSA )和模板DNA 等6 种因素5 个水平进行筛选和优化。确定甜槠ISSR-PCR 最适宜反应体系为:10 L 反应体中, 1 Taq 酶配套缓冲液(10 mmolL-1 Tris-HCl , pH 9.0 , 50 mmolL-1 KCl , 10 gL-1 TritonX-100), 1.7 mmolL-1 Mg 2+ , 0.25 mmolL-1 dNTP , 8.34 nkat Taq DNA 聚合酶, 1.5 gL-1牛血清白蛋白, 6 pmol 引物, 12 ng 模板DNA 。甜槠扩增时引物UBC846 的最适退火温度为56.3 ℃。图2 表1 参23
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| 关 键 词： | 植物学 ISSR-PCR 正交设计 0优化 甜槠 |
| 收稿时间： | 2005-10-31 |
Optimization of ISSR-PCR reaction system of Castanopsis eyrei by orthogonal design |
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| ZHANG Wen-biao,JIN Ze-xin,LI Jun-min,PAN Guan-qiong. Optimization of ISSR-PCR reaction system of Castanopsis eyrei by orthogonal design[J]. Journal of Zhejiang A&F University, 2006, 23(5): 516-520 |
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| Authors: | ZHANG Wen-biao JIN Ze-xin LI Jun-min PAN Guan-qiong |
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| Affiliation: | 1.Key Laboratory of the Three Gorge Reservoir Regions Eco-Environment of Ministry of Education, Southwest China Normal University , Chongqing 400715 , China;2.Institute of Ecology , Taizhou University , Linhai 317000 , Zhejiang , China |
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| Abstract: | Obtaining the stable and repeatable ISSR-PCR amplification result is the basic work to study genetic diversity of Castanopsis eyrei .The ISSR-PCR amplification system was optimized by using orthogonal design in six factors and five levels (Mg2 + , dNTP , primer , Taq DNA polymerase , bovine serum albumin and template DNA).The suitable reaction conditions of ISSR-PCR were shown as follows :1 Taq buffer (10 mmolL-1 Tris-HCl , pH 9.0 , 50 mmolL-1 KCl , 10 gL-1 TritonX-100), 1.7 mmolL-1 Mg 2+ , 0.25 mmolL-1 dNTP , 8.34 nkat Taq DNA polymerase , 1.5 gL-1 bovine serum albumin , 6 pmol primer , 12 ng template DNA in 10 L reaction volume .The optimal annealing temperature for primer UBC 846 was 56.3 ℃.[ Ch , 2 fig .1 tab .23 ref .] |
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