核酸的分离.PPT

课程简介 教学要求 通过教学，要求学生掌握分子生物学与基因工程的基本理论，巩固所学的理论知识； 使学生了解科研工作的基本思路，学会如何设计实验，如何分析实验，培养分析问题和解决问题的能力； 培养和训练学生的基本实验技能，培养学生的独立工作能力和创造能力。 掌握基因重组的基本过程，如：核酸DNA、RNA和质粒DNA提取、酶切、连接、转化及重组子的酶切鉴定等技术。 掌握外源基因的表达技术和蛋白质电泳分析技术，蛋白质提取、分离和纯化技术。 掌握真核生物基因组DNA的提取的基本原理。 提取高质量基因组DNA的主要用途： PCR扩增的模板 用于构建基因组文库 用于Southern blot 杂交分析 DNA Extraction 从外周血提取基因组DNA的步骤 PROCEDURE OF DNA EXTRACTION FROM BLOOD.doc RNA 分离的关键因素是尽量减少RNA酶的污染！ 如何创造一个无RNase的环境 ？ 极力避免外源RNase的污染：主要来源于操作者的手、实验的器皿和试剂 尽力抑制内源性RNase的活力：主要来源于样品中的组织细胞。 Acid-guanidinium thiocyanate-phenol-chloroform RNA purification (after Chomczynski and Sacchi, 1987) N.B: Care must be taken when handling solutions containing high concentrations of guanidine salts due to its chaotropic nature. As with other procedures involving RNA, gloves should be worn at all times to avoid contamination of samples with ribonucleases. This method describes preparation from small quantities (~ 50mg) of tissue. Appropriate scaling of the volumes involved can be performed to accomodate larger quantities. You will need: Guanidine isothiocyanate (Sigma)1M sodium citrate, pH 7 (DEPC-treated, autoclaved)Sarcosyl (Na-lauryl sarcosine, Sigma)b-mercaptoethanol (Sigma)2M sodium acetate, pH4 (DEPC-treated, autoclaved)3M sodium acetate, 100mM magnesium acetate, pH 5.2Absolute ethanolPropan-2-ol (isopropanol)70% ethanol (made with DEPC-treated, autoclaved water)0.5% SDS (made with sterile, DEPC-treated water) Solution D - 4M guanidine isothiocyanate, 25mM sodium citrate, pH7, 0.5% sarcosyl, 100mM b-mercaptoethanol. Solution D can be made and stored at 4°C without b-mercaptoethanol for several months. b-mercaptoethanol should be added to 100mM immediately prior to use. b-mercaptoethanol is used to prevent the reformation of the intra-molecular disulphide bridges , one of the reasons for the extreme stability of ribonucleases. 1) Tissue is homogenized as rapidly as possible, at 4°C, in solution D (500ul per 50mg tissue) with an eppendorf pestle homogen