实蝇RAPD反应体系的构建与优化

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

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,MgCl2浓度为2.0mM;四种dNTP浓度各为0.2mM;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。

The factors influencing RAPD analysis, including the concentration of DNA template, Mg^2＋, 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 25pA reaction solution, contained 80ng DNA template, 2.0mMMg^2＋, 0.2 mMdNTP, 0.5μMprimers, 1U Taq polymerase. The ampli- fication program was devised： 94℃ for 3rain, denaturing at 94℃ for 45see, primer annealing at 36℃ for lmin, extension at 72℃ for 2min, 40cyclesm, at last extension at 72℃ for 10min.