【摘要】 以粗壮女贞嫩芽为材料,对粗壮女贞RAPD分析中一些重要影响因素,包括模板DNA浓度、dNTPs浓度、Mg2+浓度、Taq酶用量、退火温度、延伸时间以及循环次数等因子进行了比较系统的摸索和优化.建立了适合粗壮女贞RAP-PCRD分析的优化反应体系:即25μl反应体系中Mg2+浓度为2.5mM,dNTPs浓度为200μM,Taq酶用量为2.0U,引物浓度为10pmol,模板浓度为80ng.PCR扩增程序为:94℃预变性4min,然后按94℃变性30s,37℃退火45s,72℃延伸120s,进行40个循环,最后于72℃延伸10min.以该优化的RAPD条件进行重复实验,其实验结果重现性良好.更多还原

【Abstract】 Using the young buds as the test material,several important factors in RAPD analysis of Ligustrum robustum(Roxb) Bl were systematically studied and optimized,which include the concentration of DNA template,dNTPs,Mg2+ and Taq polymerase,as well as the annealing temperate,the elongation time and the number of thermal cycle.An optimal RAPD-PCR experimental system for L.robustum was established.The optimal experimental conditions were set as the follows: in 25μl reaction system,buffer 2.5μl 10×PCR,Mg2+ 2.5mM,dNTPs 200μm,Taq polymerase 2.0U,random primers 10pmol and DNA template 80ng.The amplification program of the optimized PCR was: At first pre-denaturing at 94℃ for 4min,then 40 cycles of denaturing at 94 ℃ for 30s;annealing at 37 ℃ for 45s;extension at 72 ℃ for 120s;at last extension at 72 ℃ for 10 min.The amplification productions were stored at 4℃.A high reproducibility was obtained under the optimized experimental conditions.更多还原