陈羡德, 陈礼光, 陈珺, 罗增炎, 张志欣, 郑郁善. 苦楝RAPD反应体系的优化[J]. 西南林业大学学报, 2008, 28(1): 43-47. DOI: 10.11929/j.issn.2095-1914.2008.01.009
引用本文: 陈羡德, 陈礼光, 陈珺, 罗增炎, 张志欣, 郑郁善. 苦楝RAPD反应体系的优化[J]. 西南林业大学学报, 2008, 28(1): 43-47. DOI: 10.11929/j.issn.2095-1914.2008.01.009
CHEN Xian-de, CHEN Li-guang, CHEN Jun, LUO Zeng-yan, ZHANG Zhi-xin, ZHENG Yu-shan. Optimization of Melia azedarach RAPD Reaction System[J]. Journal of Southwest Forestry University, 2008, 28(1): 43-47. DOI: 10.11929/j.issn.2095-1914.2008.01.009
Citation: CHEN Xian-de, CHEN Li-guang, CHEN Jun, LUO Zeng-yan, ZHANG Zhi-xin, ZHENG Yu-shan. Optimization of Melia azedarach RAPD Reaction System[J]. Journal of Southwest Forestry University, 2008, 28(1): 43-47. DOI: 10.11929/j.issn.2095-1914.2008.01.009

苦楝RAPD反应体系的优化

Optimization of Melia azedarach RAPD Reaction System

  • 摘要: 以苦楝叶片提取的基因组DNA为材料,通过单因素多水平梯度试验,筛选DNA模板,Mg2+,Taq DNA聚合酶,dNTPs和随机引物的浓度及用量,建立苦楝RAPD技术分析体系.结果表明:当基因组DNA浓度为60 ng/μL,镁离子浓度为3.0 mmol/L,dNTP浓度为0.25 mmol/L,引物浓度为0.30μmol/L,Taq DNA聚合酶用量为1 U/20μL,反应体系总体积20μL时,出现可辨认的清晰谱带.其扩增程序为:94℃预变性2 min;然后38个循环(94℃变性30 s,37℃退火1 min,72℃延伸80 s);最后72℃延伸8 min,4℃保存.

     

    Abstract: Taking genomic DNA extracted from Melia azedarach foliage as experimental material,a RAPD analysis system was established by means of single factor multi-level gradient experiment to select suitable concentration and dosage for template,Mg2+,Taq DNA polymerase,dNTPs,and the random primers.The results showed that: Clear electrophoresis bands could be obtained with 60 ng/μL concentration of genomic DNA,3.0 mmol/L of Mg2+,0.25 mmol/L of dNTP,0.30 μmol/L of primer,1 U/(20 μL) of Taq DNA polymerase and when the total reaction volume was 20 μL.The optimum RAPD program was 2 min predenaturation at 94 ℃,followed by 38 reaction cycles,8 min extension at 72 ℃ and final storage at 4 ℃.Each reaction cycle included 30 sec of denaturation at 94 ℃,1 min annealing at 37 ℃ and 80 sec extension at 72 ℃.

     

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