以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、Taq DNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25舻LPcR反应体系,10×bufier2.5μL;dNTP0.24mmol/L;MgCl2 2.0mmol/L;Taq DNA聚合酶1U:DNA模板45ng;引物30ng。扩增程序为:94℃预变性lmin45 s、94℃变性30 s、35℃退火1 min30 s、72℃延伸2 min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。
By using an improved SDS method, we obtained high qualified genomic DNA of C, omphocerus sibiricus (L.). The effects of several parameters (contents of dNTP, Mg^2+ , Taq DNA polymerase and template DNA) on amplification were examined. The optimal reaction system of RAPD of G. sibiricus was determined as followed: 0.24 mmol/L dNTP, 2.0 mmol/L Mg2. , 1.0 U Taq DNA polymerase, 45 ng template DNA and 30 ng primer in total 25 μL reaction volume. Modified thermal profile consisted of an initial denaturation step at 94℃ for 1 rain and 45 s, followed by 45 cycles of 94℃ for30 s, 35℃ 1 min and 30 s and 72℃ for 2 min, and a final exposure to 72℃ for 10 min.