蓖麻RAPD-PCR反应体系的正交优化

Optimization of RAPD-PCR System with Orthogonal Design in Castor

以东北蓖麻为材料建立蓖麻RAPD反应优化体系,用于蓖麻遗传多样性分析。用CTAB提取蓖麻基因组DNA,采用正交试验对影响蓖麻RAPD-PCR扩增的反应组分浓度进行优化。结果表明:最佳的蓖麻RAPD-PCR反应体系(25μl)中含:10×buffer2.5μl,模板DNA4ng/μl,dNTP0.4mmol/L,引物0.32μmol/L及Taq酶0.1U/μl。扩增程序为:94℃预变性2min;94℃变性30s,35℃退火30s,72℃延伸1min20s;40个循环;72℃延伸5min。通过正交体系优化,获得了较优的蓖麻RAPD-PCR反应体系,为蓖麻RAPD分子标记提供了理论基础。

The optimum RAPD system of castor which was used to analyze the genetic variety of castor was established by the material of the Northeast castor. Genomic DNA of castor were extracted by CTAB method. Factors in the system were studied in order to establish the optimum RAPD system of castor with orthogonal design. Results indication： A total volume of 25μ1 RAPD-PCR system consisted of 2.5μ110×buffer, 4ng/μl template DNA, 0.4mmol/L dNTP, 0.32μmol/L primer and 0.1U Taq DNA polymerase was the optimum condition.Amplification Program initial denaturing at 94℃ for 2rains, denaturing at 94℃for 30s, annealing at 35℃ for 30s, extension at 72℃ for 1.4mins, 40cycles; final extend at 72℃ for 5mins. Through the optimizing of orthogonal system, a better RAPD-PCR reaction system of castor was retained ,which established the theoretical base for the RAPD molecular label of castor.