冬瓜和节瓜DNA提取及RAPD反应体系优化

DNA Extraction and Optimization of RAPD Reaction in Wax Gourd and Chieh-qua

以8份冬瓜和节瓜为材料,采用改良CTAB法提取基因组DNA,采用正交试验设计,对冬瓜和节瓜RAPD条件进行了优化,建立了最佳反应体系:25μL反应体系中含1×buffer,模板DNA、Mg2+、dNTPs、引物和Taq酶的浓度分别为20 ng、2.0mmol/L、0.24 mmol/L、0.3μmol/L和1.0 U。PCR扩增程序为:94℃预变性5 min;94℃变性45 s,36.9℃退火45 s,72℃延伸1.5min,共40个循环;72℃延伸10 min,12℃保存。

DNA of 8 Wax gourd （ Ben incasa hispida Cogn. ） and Chieh-qua （ Benincasa hispida Cogn. var. chieh-qua How. ） were extracted with modified CTAB method. Using the orthogonal experimental design, RAPD conditions of Wax gourd and Chieh-qua were optimized and the best reaction system was established. 1 x buffer solution was in the 25 μL reaction systems, and the concentration of template DNA, Mg2＋, dNTPs, primer and Taq enzyme were 20 ng, 2.0 mmol/L, 0.24 mmol/L, 0.3 μmol/L and 1.0 U, respectively. The optimal PCR amplification program was as follows ： predenaturation at 94℃ for 5 minutes, denaturation at 94℃ for 45 seconds, annealing at 36.9℃ for 45 seconds, extension at 72℃ for 1.5 minutes. The cycle repeated 40 times. Extension was at 72℃ for 10 minutes and stored at 12℃.