| 寒兰基因组DNA ISSR-PCR反应条件的优化 |
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| 引用本文： | 孙小琴,李恩香,贾文杰,彭德镇,孔令杰,杨柏云.寒兰基因组DNA ISSR-PCR反应条件的优化[J].安徽农业科学,2009,37(29):14044-14046. |
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| 作者姓名： | 孙小琴 李恩香 贾文杰 彭德镇 孔令杰 杨柏云 |
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| 作者单位： | 南昌大学生命科学学院,江西南昌,330031;南昌大学生命科学学院,江西南昌,330031;南昌大学生命科学学院,江西南昌,330031;南昌大学生命科学学院,江西南昌,330031;南昌大学生命科学学院,江西南昌,330031;南昌大学生命科学学院,江西南昌,330031 |
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| 基金项目： | 江西省教育厅科学技术研究项目 |
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| 摘 要： | 以改良的CTAB法提取的寒兰（Cymbidium kanran Makino）基因组DNA为模板，通过单因子试验建立最适的寒兰的ISS-PCR反应体系。结果表明，适宜寒兰ISSR-PCR反应体系的扩增条件为：25 μl PCR 反应体积中，1×PCR buffer，2.0 mmol/L MgCl2，300 ng 模板 DNA，200 μmol/L dNTP，1.40 U Taq DNA 聚合酶，0.4 μmol/L 引物。最佳扩增程序为：94 ℃预变性 5 min，然后进行40个循环：94 ℃ 变性 30 s，复性温度根据各引物的Tm值略低1～2 ℃，30 s，72 ℃ 延伸 50 s，循环结束后 72 ℃ 延伸7 min。
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| 关 键 词： | 寒兰 ISSR-PCR反应 遗传多样性 |
Optimization of ISSR-PCR Reaction Conditions for Genomic DNA from Cymbidium kanran Makino |
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| SUN Xiao-qin et al.Optimization of ISSR-PCR Reaction Conditions for Genomic DNA from Cymbidium kanran Makino[J].Journal of Anhui Agricultural Sciences,2009,37(29):14044-14046. |
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| Authors: | SUN Xiao-qin |
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| Institution: | SUN Xiao-qin et al (School of Life Science,Nanchang University,Nanchang,Jiangxi 330031) |
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| Abstract: | The genomic DNA of C.kanran was used as template,which was extracted by improved CTAB.The suitable reaction ISSR-PCR system of C.kanran was established by studying the influence of the main components in the ISSR reaction system.The most suitable ISSR-PCR reaction system and amplified procedure for C.kanran was 25 μl amplification reactions system which contained 1×PCR buffer,2.0 mmol/L MgCl2,300 ng template DNA,200 μmol/L dNTPs,1.40 U Taq DNA polymerase,0.4 μmol/L primer.The optimal amplified procedure was as follows: a pre-denaturing for 5 min at 94 ℃,40 cycles of denaturing for 30 s at 94 ℃,annealing for 30 s at the temperature due to 1-2 ℃ lower than the Tm of different primer and extension for 50 s at 72 ℃.At last,the DNA in the reactions system was extended for 7 min at 72 ℃. |
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| Keywords: | Cymbidium kanran ISSR-PCR reaction Genetic diversity |
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