苦楝RAPD反应体系的优化

Optimization of Melia azedarach RAPD Reaction System

以苦楝叶片提取的基因组DNA为材料,通过单因素多水平梯度试验,筛选DNA模板,Mg2+,Taq DNA聚合酶,dNTPs和随机引物的浓度及用量,建立苦楝RAPD技术分析体系.结果表明:当基因组DNA浓度为60 ng/μL,镁离子浓度为3.0 mmol/L,dNTP浓度为0.25 mmol/L,引物浓度为0.30μmol/L,Taq DNA聚合酶用量为1 U/20μL,反应体系总体积20μL时,出现可辨认的清晰谱带.其扩增程序为:94℃预变性2 m in;然后38个循环(94℃变性30 s,37℃退火1 m in,72℃延伸80 s);最后72℃延伸8 m in,4℃保存.

Taking genomic DNA extracted from Melia azedarach foliage as experimental material, a RAPD analysis system was established by means of single factor multi - level gradient experiment to select suitable concentration and dosage for template, Mg^2＋ , Taq DNA polymerase, dNTPs, and the random primers. The results showed that： Clear electrophoresis bands could be obtained with 60 ng/μL concentration of genomic DNA, 3.0 mmol/L of Mg^2＋ , 0.25 mmol/L of dNTP, 0.30 μmol/L of primer, 1 U/（20 μL） of Taq DNA polymerase and when the total reaction volume was 20 μL. The optimum RAPD program was 2 min predenaturation at 94℃, followed by 38 reaction cycles, 8 min extension at 72 ℃ and final storage at 4 ℃. Each reaction cycle included 30 sec of denaturation at 94 ℃, 1 min annealing at 37℃ and 80 sec extension at 72 ℃.