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.
Using the young buds as the test material, several important factors in RAPD analysis of Ligustrum robustum （Roxb） B1 were systematically studied and optimized, which include the concentration of DNA template, dNTPs, Mg^2 ＋ 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 condi- tions were set as the follows： in 25μl reaction system, buffer 2.5μl 10 × PCR, Mg^2 ＋ 2.5mM, dNTPs 200μm, Taq pelymerase 2.0U, random primers 10pmol and DNA template 80ng. The amplification program of the optimized PCR was： At first pre - denaturing at 94℃ for 4rain, 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.
出处
《贵州科学》
2007年第2期56-64,共9页
Guizhou Science
基金
贵州省自然科学基金资助项目
批准文号:黔科合J字〔2005〕2033号
关键词
苦丁茶
粗壮女贞
RAPD
反应体系
kudingcha
Ligustrum robustum （Rox） B1.
RAPD
reaction system
