After many unsuccessful attempts to obtain biologically active mRNAs from dry seeds of Davidia involucrata using available protocols,an effective procedure modified from Vicient and Delseny (1999) is developed.This me...After many unsuccessful attempts to obtain biologically active mRNAs from dry seeds of Davidia involucrata using available protocols,an effective procedure modified from Vicient and Delseny (1999) is developed.This method is based on the use of 8mol/L LiCl in the extraction buffer followed by phenol extractions.The differences with respect to the original method are the inclusions of 2% soluble PVP (10000Da) and 10 mmol/L 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.The author 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 and 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.展开更多
文摘After many unsuccessful attempts to obtain biologically active mRNAs from dry seeds of Davidia involucrata using available protocols,an effective procedure modified from Vicient and Delseny (1999) is developed.This method is based on the use of 8mol/L LiCl in the extraction buffer followed by phenol extractions.The differences with respect to the original method are the inclusions of 2% soluble PVP (10000Da) and 10 mmol/L 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.The author 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 and 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.
