北五味子RAPD-PCR反应条件优化

Optimization of RAPD-PCR reaction system of Schisarutra chinesis(Turcz.) Baill

以采自吉林省汪清地区野生北五味子叶片为材料,提取其基因组DNA,并以此为模板进行北五味子RAPD-PCR反应条件的优化.结果表明,PCR反应混合液为:25μL PCR反应体系中加入20 ng模板DNA,200μmol/L dNTPs,0.3μmol/L引物,2.0 mmol/L Mg2+,1 UTaqDNA聚合酶,2.5μL 10×Buffer;PCR反应程序为:94℃预变性5 min后,在94℃变性1 min,40℃退火1 min,72℃延伸2 min,共40个循环后,在72℃下最后延伸5 min时,在不同引物中均扩增出清晰而稳定的DNA电泳谱带.

The optimal RAPD-PCR reaction system of Schisarutra chinesis （Turcz.） Baill was established with genomic DNA extracted from fresh leaves. The results indicated that in total 25μL volumes of PCR reaction, contain 20 ng of DNA, 200 μmol/L of dNTPs, 0.3 μmol/L of primers, 2.0 mmol/L of Mg^2＋ , 1 unit of TaqDNA polymerase, 2.5μL of 10 ）〈 Buffer ; and the PCR program is after predenaturing at 94℃ for 5 min, followed by 40 cycles of denaturing at 94 ℃ for 1 min,annealing at 40 ℃ for 1 min,extension at 72℃ for 2 min,and final extension at 72℃ for 5 min.