《哺乳动物基因组DNA的提取Southern杂交》-毕业论文（设计）.docVIP

小鼠基因组DNA的提取及Southern杂交 12 - 分子生物学实验报告 哺乳动物基因组DNA的提取 Southern杂交 组 别：二组 组 员： 摘要:本实验在无液氮的情况下，制备鼠肝基因组DNA。并将产物DNA做Southern杂交实验。Southern 杂交是分析基因结构或检测DNA中是否含有特定序列的有效技术之一, 1975年由Southern E M 首创, 取其姓氏而命名。Southern杂交中探针有放射性标记和非放射性标记系统，本实验采用非放射性标记系统中的地高辛标记系统。本实验先将凝胶电泳分离的小鼠DNA片段转移到尼龙膜上，然后用地高辛标记的碱性磷酸酶基因探针与之杂交，再通过显色反应来判断所提取的小鼠基因组DNA中是否含有与碱性磷酸酶基因同源的片段以及该同源片段的长度。Southern杂交的转膜过程通过“夹心饼”式结构，即“滤纸-尼龙膜-凝胶-滤纸”完成。在Southern杂交之前要进行预杂交，以此将杂交膜上的非特异性DNA结合位点封闭，减少与探针的非特异性吸附降低杂交结果的本底。在鼠肝基因组DNA的制备过程中，要尽量避免DNA酶的污染，特别注意动作温和，减少对DNA 的机械损伤。 关键词：小鼠基因组DNA提取，Southern杂交 Abstract：In this study，the liver genomic DNA of rat was prepared without the liquid nitrogen. The DNA product was used to do Southern hybridization. Southern hybridization is one of the effective technologies of analyzing the gene structure and detecting specific DNA sequences, which was pioneered in 1975 by Southern EM and named after him. There are two kinds of probes in Southern blot that includes radio labeled system and non-radioactive system. In present study, the Digoxigenin-labeled system of non-radioactive labeling system was applied. In this study, transferring the mice DNA fragments from gel electrophoresis to nylon membrane first and hybriding it with Digxigenin-labeled probe of alkaline phosphates’ gene. At last, determine whether the genomic DNA extracted from mice has homologous fragments with alkaline phosphates’ gene and the length of the homologous fragment by color reaction. The transfer film process of Southern blot was finished by puffs structure--the filter paper -nylon film -gel - filter. In the pre-hybridization before hybridization, the blots as a non-specific DNA binding sites were closed, which can reduce non-specific adsorption of the probe hybridization and the background. Avoiding the pollution of DNA enzymes, and taking gentle action when preparing the liver genomic DNA of rat so as to reduce the mechanical damage of DNA. Key words Mouse genomic DNA extraction, Southern blotting。 一、引言 1、鼠肝基因组DNA的