一种木本植物RNA和DNA的简捷提取方法

A Convenient Method of RNA and DNA Extraction from Woody Plant

将尾叶桉(Eucalyptus urophylla)叶片在液氮中研磨成粉,先用含山梨醇、聚乙烯吡咯烷酮(PVP)、牛血清白蛋白、聚乙二醇和亚精胺的提取缓冲液使总核酸沉淀,同时去除了大部分次生代谢物质。然后用山梨醇、月桂酰肌氨酸钠进一步裂解细胞,释放核酸。再用CTAB与核酸结合成复合物溶于高盐溶液。用氯仿/异戊醇除蛋白质后,离心得含总核酸的上清液。运用钙盐分步沉淀法,先在上清液中加入1/10体积的10%氯化钙溶液,使DNA与Ca2+结合成DNA钙盐。向溶液中加入1/5体积的乙醇,使DNA钙盐形成沉淀析出,离心得DNA后,再增大乙醇浓度使RNA钙盐沉淀。得到的DNA和RNA用非变性琼脂糖凝胶进行质量检测,可得完整的RNA和DNA条带。此法经济、快捷,可同时得到DNA和RNA。

Using fresh leaf of E. urophylla as material, RNA and DNA were extracted and separated at the same time. Firstly, sorbitol, polyvinylpyrrolidone, bovine serum albumin, polyethylene glycol and spermidine were used to precipitate total nucleic acid while most of secondary metabolites were removed. Secondly, sorbitol and sodium lauroyl sarcosine were used to schizolysis cells further. Thirdly,CTAB and nucleic acid formed soluble complex at high-salt solution. After removing proteins by chloroform, DNA was precipitated by adding one tenth volume of 10% calcium chloride solution and one fifth volume of ethanol. Lastly, RNA was precipitated by increasing the content of ethanol. The result of electrophoresis at nondenaturing agarose gel showed both RNA and DNA were high quality. The method was economical, simple and fast. And RNA and DNA were extracted at the same time.