摘要
为了建立广霍香优化的ISSR-PCR反应体系,首先通过单因子试验选定其各影响因子比较适宜的浓度范围,再利用正交试验设计的方法,对影响广藿香ISSR-PCR反应的5种因素4水平进行优化试验。结果表明:广藿香ISSR-PCR的优化反应体系最终确定为:在25μL反应体系中,含DNA模板40 ng,Mg2+浓度为2.5 mmol.L-1,引物浓度为0.3μmol.L-1,TaqDNA聚合酶用量为1.5 U,dNTPs浓度为150μmol.L-1。PCR扩增程序为:94℃预变性5 min,然后按94℃变性45 s,52.7℃退火45 s,72℃延伸90 s,进行40个循环,最后72℃延伸7 min,4℃保存。
In order to establish the optimized ISSR-PCR experiment system for Pogostemon cablin,the single factor experiments were used to detect the suitable concentration ranges of the different influential factors,and then the orthogonal design was performed to optimize 5 factors（Mg^2＋,dNTPs,primer,Taq polymerase,DNA template） at 4 levels,which affect ISSR-PCR amplification system of Pogostemon cablin.The results indicated that a suitable ISSR-PCR reaction system established： 10× buffer 2.5 μL,40 ng DNA template,2.5 mmo·L^-1MgCl2,0.3 μmo·L^-1primers,1.5 U Taq polymerase,150 μmo·L^-1dNTPs were contained in 25 μL reaction solution.The optimized amplification program was that predenaturing at 94 ℃ for 5 min,then denaturing at 94 ℃ for 45 s,primer annealing at 52.7 ℃ for 45 s,extension at 72 ℃ for 90 s,for 40 cycles,at last extension at 72 ℃ for 7 min,and the products were stored at 4 ℃.
出处
《热带生物学报》
2011年第1期35-41,共7页
Journal of Tropical Biology
