Southern印迹先将DNA经限制性内切酶消化成一系列片段.ppt

动物组织(2mg)用液氮研磨后,加入500ul lysis buffer , 振荡至彻底悬浮. 加入20ul蛋白酶K溶液(20mg/ml),混匀.55℃放置4-16小时,直到组织溶解. 加入等体积的酚：氯仿：异戊醇（25：24：1） ,充分颠倒混匀. 14000rpm离心5min，上清转移置另一干净离心管中 重复抽提一次 加2倍体积无水乙醇颠倒混合沉淀DNA。 14000rpm离心10min ，DNA沉淀形成白色絮状物，去上清。 70%乙醇洗沉淀两次，室温干燥 加入50-100ul TE，55℃ 3-4小时溶解 DNA提取操作步骤 Southern blot 动物生物技术系 Southern印迹是将DNA片断从电泳凝胶上直接转移至膜支持物（如硝酸纤维素膜、尼龙膜）上，使DNA片断固定的技术。 Southern印迹 先将DNA经限制性内切酶消化成一系列片段， 进行琼脂糖凝胶电泳，各片段因分子量不同而彼此分开， 然后经碱处理凝胶，使DNA的片段被变性、中和并通过毛细作用在高盐缓冲液中在原位将单链核酸转印到硝酸纤维膜上，烘干、固定。 分子生物学中使用的标记方法 杂交研究中的核酸探针 核酸杂交探针使用的标记物 随机引物的探针标记 DNA的限制性酶切 Southern 印记 DNA分子 限制片段 限制性酶切割 琼脂糖电泳 转移至硝酸纤维素膜上 与放射性标记DNA探针杂交 放射自显影 带有DNA片段的凝胶 凝胶 滤膜 用缓冲液转移DNA 吸附有DNA片段的膜 Southern 印迹法 Albert Lasker Award for Clinical Medical Research Alec John Jeffreys and Edwin M. SouthernAlec John Jeffreys and Edwin M. Southern for development of two powerful technologies - Southern hybridization and DNA fingerprinting - that together revolutionized human genetics and forensic diagnostics. Alec John JeffreysUniversity of Leicester (UK) Edwin SouthernUniversity of Oxford (UK) Southern 印迹法的历史背景 Southern Hybridization An agarose gel is prepared and run according to the standard procedures, then it is stained and photographed to have a record of all the DNA fragments. The gel is prepared by first denaturing, then neutralizing, the DNA fragments in the gel Southern hybridization has many steps and usually requires more than one day (or lab period) to complete. This procedure can be broken down into several sections Preparing the gel After neutralization, the gel is carefully measured. Once the gel dimensions are known, we can begin to assemble the materials needed for Southern transfer. The nylon membrane is cut to the size of the gel. The membrane itself is sandwiched between two sheets of protective paper. However, wear gloves when working with the membrane to further protect it from oil and dirt.? Two pieces of 3 mm filter paper are also cut to the size of the gel. Students should also prepare the stack of paper towels