葛属植物ISSR-PCR扩增条件的正交优化

利用正交试验设计法L9(34),从引物浓度、TaqDNA聚合酶浓度、Mg2+浓度和dNTP浓度4种因素3个水平,对葛资源ISSR-PCR反应体系进行优化分析,并在此基础上对PCR反应的退火温度进行梯度筛选。结果表明,25μL最佳的葛资源ISSR-PCR反应体系是:1×PCR buffer、0.20 mmol/L dNTP、0.4μmol/L引物、2.5 mmol/L Mg2+和0.5 U TaqDNA聚合酶,引物UBC809的最佳退火温度为57.9℃。

Orthogonal optimization of ISSR-PCR reaction system in Pueraria

In this study, orthogonal experimental design method L_9 (3~4)was used, from the concentration of primers, Taq DNA polymerase concentrations, Mg~(2+) concentration and dNTP concentrations of four kinds of factors in three levels, ISSR-PCR reaction system of Pueraria was analysed. On this basis, the annealing temperature were proposed by gradient PCR. The results showed that, 25 μL ueraria resources ISSR-PCR reaction system was: 1×PCR buffer, 0.20 mmol/L dNTP, 0.4 μmol/L primer, 2.5 mmol/L Mg~(2+)and 0.5 U Taq DNA polymerase, the optimal annealing temperature of primer UBCS09 was 57.9℃.