论文部分内容阅读
通过单因素与中心复合设计相结合的方法建立优化红芪ISSR-PCR反应体系,并筛选引物,优化电泳时间及扩增循环数。建立的红芪ISSR-PCR反应体系为:25μL反应体系中10×PCR Buffer(Mg~(2+))3.5μL、模板DNA(30 ng/μL)2μL、PCR扩增引物2μL、Taq DNA聚合酶为1.25 U、d NTPs为2μL,dd H_2O 14.5μL,建立的扩增程序:94℃预变性5 min,开始34个循环;94℃变性30 s,后据不同退火温度的引物复性45 s,72℃延伸2 min,循环结束后72℃延伸7 min。本研究筛选出红芪扩增的引物17条;PCR反应产物电泳时间为120 min,最佳循环数为34,建立了稳定的体系,为进一步研究红芪的遗传多样性及遗传结构提供了帮助。
Through the combination of single factor and central composite design method, ISR-PCR reaction system was optimized and the primers were screened to optimize the electrophoresis time and the number of amplification cycles. The established ISSR-PCR reaction system of Radix Astragali was: 3.5 μL of 10 × PCR Buffer (Mg 2+), 2 μL of template DNA (30 ng / μL), 2 μL of PCR amplification primer, Taq DNA polymerase Was 1.25 U, dNTPs was 2μL, and dd H_2O was 14.5μL. The amplification program was established: pre-denaturation at 94 ° C for 5 min and start of 34 cycles; denaturation at 94 ° C for 30 s followed by annealing at different annealing temperatures for 45 s, 72 ℃ for 2 min, 72 ℃ for 7 min after the end of the cycle. In this study, 17 primers were screened for the amplification of Radix Hedysari. The electrophoresis time of the PCR reaction was 120 min and the optimal number of cycles was 34. A stable system was established for further study of the genetic diversity and genetic structure of Radix Hedysari .