正交设计优化黑褐苔草ISSR-PCR反应体系

Optimizing ISSR-PCR System of Carex atrofusca Using Orthogonal Design

采用正交设计对黑褐苔草(Carex atro fusca)ISSR-PCR反应的Mg^(2+)、Taq DNA聚合酶、dNTP、引物和DNA模板进行优化,以期建立最佳反应条件并筛选退火温度。结果表明:黑褐苔草20μL ISSR-PCR最佳反应体系为:1.80μmol·L^(-1)Mg^(2+)、0.60 U Taq DNA聚合酶、0.125μmol·L^(-1)dNTP、0.3μmol·L^(-1)Primer和50 ng DNA模板;引物UBC807适宜退火温度为55℃。该体系能在不同个体中扩增出清晰的、稳定性好的条带。

An orthogonal design was used to optimize the ISSR PCR system of Carex atrofusca at four levels of five factors （Mg2＋,Taq DNA polymerase,dNTP,primer and DNA template）.Then,based on the optimal ISSR PCR system,the annealing temperature was selected.The optimal PCR （20 μL） mixture contained 1.80 mmol · L-1 Mg2＋,0.60 U Taq DNA polymerase,0.125 mmol · L-1 dNTP,0.3 μmol · L-1 primer and 50 ng template DNA.The suitable annealing temperature of primer UBC807 was 55℃.With the optimal system of ISSR PCR,clear and steady bands were obtained in different individuals of C.atrofusca.