硅胶干燥罗布麻叶片DNA提取及RAPD反应体系建立

摘要

[目的]得到适用于罗布麻RAPD的最优体系.[方法]以伊犁地区新源县罗布麻硅胶干燥叶片为材料,采用TIANGEN试剂盒提取罗布麻基因组DNA,得到满足RAPD(Random amplified polymorphic DNA)分析的罗布麻基因组DNA.[结果]通过单因素优化实验,得到罗布麻RAPD分析的最佳反应体系为:Mg2+ (2.0mmol/L)、dNTPs(0.5 mmol./L)、primer(0.4 mmol/L)、DNA(1μL)、Tap酶取1μL、10×buffer取1μL,总反应体系为10 μL;扩增程序为:在94℃下预变性,然后进行42个循环(94℃变性30 s、38℃退火30 s、72℃延伸2min),最后在72℃延伸7 min.[结论]提取的罗布麻总DNA均有较高的质量,适合RAPD分析；Tiangen植物基因组DNA提取试剂盒对罗布麻基因组DNA有着良好的提取效果；对罗布麻RAPD-PCR中Mg2+、dNTPs、DNA聚合酶和引物浓度进行优化,较为理想的各因子用量和扩增程序为:Mg2+(2.0 mmol/L)、dNTPs(0.5mmol/L)、primer(0.4 mmol/L)、Tap酶(0.5 U/μL)、DNA(40 ng),总反应体系为10 μL;扩增程序为:在94℃下预变性5 min,然后进行42个循环(94℃变性30 s、38℃退火30 s、72℃延伸2 min),最后在72℃延伸7min.%[Objective] The purpose of this project is to obtain the optimal RAPD reaction system that might apply to apocynum. [Method] Apocynum in Xinyuan County is used as experimental materials and the genomic DNA was extracted from young leaves of Apocynum by means of Tiangen Kit until sufficient RAPD (Random amplified polymorphic DNA) is got to carry out analysis of Apocynum. [Result] Through single factor experiments, The best RAPD reaction system optimized was; Mg2+ (2.0 mmol/L) , dNTPs(0. 5 mmol/ L), primer(0.4 mmol/L) 、DNA(0. 3 U), Taq DNA polymerase 1 L, 10 × buffer 1 L,10 L PCR solution;The RAPD PCR was programmed by a cycle for 5 min at 941, followed by 42 cycles for 30s at 94℃, 30s at 38℃: and 2 min at 72℃, and finally by a cycle for 2 min at 72℃. [ Conclusion ] The obtained DNA is of high quality, which applies to RAPD analysis.