[关键词]
[摘要]
以兰州百合鳞片和叶片为试材,采用改良CTAB法获得了高质量的基因组DNA。为优化兰州百合RAPD-PCR反应体系,利用正交试验设计,对兰州百合RAPD-PCR反应体系中的5种主要因素进行4水平共计16个组合设计,并选用3种反应程序,通过琼脂糖凝胶电泳结果分析,最终获得最佳反应体系为:20 uL反应体系中含30 ng模板DNA、0.5 μmol/L随机引物、0.2 mmol/L dNTPs、2.25 mmol/L MgCl2, 及1.5U Taq DNA聚合酶。最佳反应程序:94 ℃预变性4 min;94 ℃变性1 min,36 ℃退火1.5 min;72 ℃延伸2 min;35个循环;最后72 ℃延伸7 min。
[Key word]
[Abstract]
High quality genomic DNA is obtained from lily bulbs and leaves using improved CTAB method. In order to get reliable and repeatable reaction system of RAPD-PCR amplification for Lanzhou lily, we optimized the RAPD-PCR reaction system at 4 levels and 5 factors by orthogonal experimental design. The optimized RAPD-PCR system is tested on 3 PCR amplification procedure. The result of polyacrylamide gel electrophpresis shows that the best reaction system is 20 ul reaction solution, which contained 30 ng template DNA, 0.5 μmol/L primers, 0.2 mmol/L dNTPs, 2.25 mmol/L MgCl2 and 1.5 U Taq DNA polymerase. The reaction procedure is as follows: pre-denaturing at 94 ℃ for 4 min, 35 cycles of denaturing at 94 ℃ for 1 min,annealing at 36 ℃ for 1.5 min and extending at 72 ℃ for 2 min, extending at 72 ℃ for 7 min after the cycles.
[中图分类号]
S644.1
[基金项目]
甘肃省农业科学院青年基金项目(2013GAAS28);甘肃省农业科学院科技创新工程学科团队项目(2015GAAS02)。