【摘要】 改进后的方法在提取缓冲液中加入了2%的PVP(分子量10000)和10mM的半胱氨酸以避免多酚类物质的氧化,减少了苯酚抽提的次数.使用该改进的方法可以从平均每克珙桐干种子中获得240μg的总RNA,这些总RNA适合用于体外反转录、Northern杂交以及cDNA文库的构建.更多还原

【Abstract】 After many unsuccessful attempts to obtain biologically active mRNAs from dry seeds of Davidia involucrata using available procedures, an effective procedure modified from Vicient and Delseny (1999) is developed. This method is based on the use of 8 M LiCl in the extraction buffer followed by phenol extractions. The differences with respect to the original method are the inclusions of 2% soluble PVP (MW 10,000, Sigma) and 10 mM cysteine in the extraction buffer to avoid oxidation of polyphenolics, the reduction in the number of phenol extractions, freezing the seeds for 10 minutes instead of using quarts. Our modifications conquer the perishing oxidation of polyphenolics, in their oxidized forms, polyphenols can covalently bind to proteins and nucleic acids; and permit the recovery of an average of 240 μg total RNA per g of the seeds that is suitable for translation in vitro, northern hybridizations, and the construction of cDNA libraries.更多还原