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中国农学通报

中国农学通报 ›› 2007, Vol. 23 ›› Issue (1): 58-058.

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实蝇RAPD反应体系的构建与优化

张长禹,孟建玉,张小亚,雷朝亮   

  • 出版日期:2007-01-05 发布日期:2007-01-05

Optimization For RAPD Reaction System in Fruit Flies (Diptera:Tephritidae)

Zhang ChangYu, Meng JianYu, Zhang XiaoYa, Lei Chaoliang   

  • Online:2007-01-05 Published:2007-01-05

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摘要： 以几种常见的实蝇为材料，研究了实蝇RAPD分析过程中的影响因素，包括模板DNA浓度、dNTP、引物、Taq酶、变性时间、循环次数、退火温度等，建立了适于实蝇RAPD分析的PCR反应体系：在25μl反应体系中，模板DNA用量为80ng，MgCl 2浓度为2.0mM；四种dNTP浓度各为0.2 mM；Taq引物浓度为0.5μM；酶用量为1U；扩增程序：先94℃预变性3min，再94℃变性45s、36℃退火1min、72℃延伸2min共40个循环，最后延伸10min。

关键词: 绞股蓝, 绞股蓝, 叶龄, 皂甙

Abstract: The factors influencing RAPD Analysis, including the concentration of DNA template, Mg2+, dNTP, primers, Taq polymerase, denaturing time, Thermal cycles and annealing temperature in fruit flies were studied. An optimal PCR system for RAPD in fruit flies has been found: in 25μl reaction solution, contained 80ng DNA template, 2.0mMMg2+, 0.2 mMdNTP, 0.5μMprimers, 1U Taq polymerase.The amplification program was devised: 94℃ for 3min; denaturing at 94℃ for 45sec, primer annealing at 36℃ for 1min, extension at 72℃ for 2min, 40cyclesm, at last extension at 72℃ for 10min.

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