黄瓜RAPD体系的优化与应用

Optimization and Application of RAPD System for Cucumber

以黄瓜为实验材料,采用改良CTAB法提取黄瓜基因组DNA进行RAPD分析,优化黄瓜RAPD体系的程序、模板、引物、dNTPs、Mg2+等参数。结果表明:黄瓜的PCR程序为94℃预变性3min;94℃变性30s,37℃复性30s,72℃延伸2min,循环40周,最后72℃延伸7min为佳;反应体系中,模板DNA的适宜浓度为2.5～5ng·μl-1,适宜的引物浓度为0.6μmol·L-1,dNTPs的适宜浓度为0.25mmol·L-1,Mg2+适宜浓度为1.875mmol·L-1。同时应用这一DNA扩增体系,进行黄瓜RAPD分析,筛选出了适用于申绿64的2条引物,最终得到了它的特异性条带。

The genomic DNA was extracte d from cotyledons of cucumber by means of advanced CTAB method.The RAPD system for cucumber was optimized including pr ogram,template,primer,dNTPs and Mg 2+ .The result showed that the most suit able program was one cycle at 94℃/3min,40cycles a t 94℃/30s,37℃/30s,72℃/2min and one cycl e at 72℃/7min.In the RAPD system of cucumber,the optional concentra tion of template DNA was 2.5～5ng·ìl-1 ,primer 0.6ìmol稬-1 ,dNTPs 0.25mmol·L-1 ,Mg 2+ 1.875mmol·L-1 .Cucumber cultivars were analyzed w ith the RAPD.Two primers which were a pplied to Shenlu 64were selected,the distinct bands of it we re also obtained .