Notopterygium and wide range of leaf Notopterygium simple sequence repeat amplification reaction system of orthogonal optimization.doc

 PAGE \* MERGEFORMAT 17 Notopterygium and wide range of leaf Notopterygium simple sequence repeat amplification reaction system of orthogonal optimization Author: Gusong JIANG Shun-yuan Tang Xue-fang Sun Hui YANG Zhi-rong Sun Qun [Abstract] Objective To explore the Notopterygium and wide-leaf Notopterygium ISSR experiment the impact of various factors in order to establish Notopterygium and wide-leaf Notopterygium range amplification of simple sequence repeat (ISSR-PCR) the best reaction. Method of orthogonal experiment design L9 (34) pairs of Notopterygium and wide leaf Notopterygium ISSR-PCR reaction system of four factors (Mg2, dNTP, primers, Taq enzyme) in the three levels of optimization experiments, PCR Results SPSS13.0 Statistics software analysis, and set the gradient PCR screening the primer annealing temperature. Results 20 μ l ISSR-PCR reaction system, the optimal concentration of each component: 2.0 mmol / L Mg2, 250 μ mol / L dNTPs, 0.50 μ mol / L primer, 1U Taq enzyme, 40 ng DNA template; template DNA 40 ~ 90 ng, different primers have different optimal annealing temperature range of 48 ~ 52 ℃ . Conclusion The establishment of the optimization system further Notopterygium and wide-leaf Notopterygium study of genetic diversity among populations as a reference. [Keywords:] Notopterygium a wide range of leaf Notopterygium simple sequence repeat amplification orthogonal experimental design Abstract: Objective The optimal ISSR-PCR reaction system for Notopergium incisum Ting and N. forbesii Boissin was constructed by studying the influence of ISSR factors.MethodsThe orthogonal design with three levels of four factors (Taq DNA polymerase, Mg2, dNTPs and primer) was used to optimize ISSR  PCR amplification reaction system of N. incisum. and N. forbesii. The data of PCR results were analysed with SPSS software, and the different annealing temperatures were also studied by gradient PCR.ResultsThe optimal ISSR-PCR reaction system contained 2.0 mmol /