Efficient isolation of high quality nucleic acids from different tissues of Taxus baccata L.
Efficient isolation of high quality nucleic acids from different tissues of Taxus baccata L.
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DOI：
10.1007/s11033-009-9607-2
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发表时间：
2010-02-01
影响因子：
2.8
通讯作者：
Mofid, Mohammad Reza
中科院分区：
文献类型：
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作者：
Kejani, Abolghasem Abbasi;Tafreshi, Sayed Ali Hosseini;Mofid, Mohammad Reza
为从不同来源的伊朗红豆杉（Taxus baccata L.）中提取高质量的DNA和RNA，建立了一种改进的、高效的方法。该方法基于添加有高水平的聚乙烯吡咯烷酮（PVP）和β-巯基乙醇的CTAB提取缓冲液，以适当地去除多糖并防止酚类化合物的氧化。通过乙醇沉淀获得的沉淀仅用氯仿：异戊醇（24：1）洗涤。因此，我们可以成功地从分离过程中消除危险的苯酚/氯仿萃取步骤。分离的核酸（DNA和RNA）的分光光度（A（260）/A（280）和A（260）/A（230）比率）和琼脂糖电泳分析均显示良好的结果。可获得平均产量为100-300 μ g/g叶、茎组织的DNA和平均产量为20-30 μ g/g细胞培养物和80-100 μ g/g叶、茎组织的总RNA。pam和pds经PCR和RT-PCR成功扩增，分别证明了DNA和RNA的完整性。
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and beta-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A(260)/A(280) and A(260)/A(230) ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100-300 mu g/g leaf and stem tissue and total RNA with an average yield of 20-30 mu g/g cell culture and 80-100 mu g/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.