An efficient and rapid method for sweetpotato genomic DNA extraction
2007
Li Qiang, Jie Qin, Liu Qingchang
Chinese. 甘薯基因组DNA传统的CTAB提取法,提取DNA质量好,但提取效率不高。为满足甘薯分子标记辅助育种、核心种质和遗传连锁图谱构建以及转基因植株检测的需要,通过对传统的CTAB法进行改进,本研究建立了一种高效快速价廉的甘薯及其近缘野生种基因组DNA提取方法,适合于PCR扩增以及AFLP分析。本方法使用1.5mL离心管和专用棒槌,整个过程转管1次,提取的DNA主带明显,片段大小一致,得率达5μg/mg叶片冻干粉。提取质量可靠,RAPD-PCR和酶切后AFLP扩增,与传统CTAB法提取DNA扩增结果没有明显差异。与传统的CTAB方法相比,该方法快速(提取过程不足30min),效率高,从10mg叶片冻干粉中一次可提取50μg左右的DNA模板,操作简便,污染降低,实用性强。
Show more [+] Less [-]English. CTAB method are popularly used for Genomic DNA extraction of Sweetpotato, but its efficiency was not high for large scale DNA isolation. In this research an efficient, rapid, and cheap method was developed for genomic DNA extraction of sweetpotato and its wild relatives by modifying CTAB method, which the DNA quality could fulfill the need of research purposes in the fields of Marker-assisted Selection (MAS), identification of the core germplasm, construction of the genetic linkage map, and detection the transgenic plants. DNA samples would be suitable for AFLP and RAPD-PCR analysis. This method employed 1.5 ml microcentrifuge tube and adaptable roller. The extracted DNA band was much more clear and the fragment size was quite sharp. The amount of DNA extraction from leaf was about 5 μg/mg freeze-dried powder. There is no obvious difference for RAPD-PCR and AFLP analysis by using different DNA templates extracted from the improving method and CTAB method. The results indicated that the protocol could be conducted finally within 30 min and about 50μ g DNA could be isolated from 10 mg freeze-dried leaf powder. Definitely the improved protocol was quite simplified one-tube transferring, which make the procedures to be operated easily.
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