哺乳动物组织基组DNA提取实验报告.docVIP

实验序号 五 实验名称 哺乳动物组织基因组DNA提取（Genomic DNA Extraction from Mammal Tissue） 实验时间 2012-5-10 实验室 生物综合实验室I 实验目的 Experimental Purpose After the experiment done, students should be able to know how to extract genomic DNA from mammal tissue, and to run an agarose gel. 1.掌握动物基因组DNA提取的操作方法。 2.掌握琼脂糖凝胶电泳的操作的方法二、实验原理 Experimental Principle In the procedure described here, the detergents SDS sodium dodecyl sulfate are added to denature proteins and solubilize lipids in membranes leading to cell lysis . The cell lysate is often treated with enzymes that hydrolyze RNA and proteins. 本实验利用去垢剂SDS 十二烷基磺酸钠 是为了使蛋白变性并溶解细胞膜中的脂质从而导致细胞裂解，而细胞裂解物又常用蛋白酶K和蛋白进行水解处理。 This is generally followed by phenol of phenol: chloroform extraction to remove the remaining proteins, which will either enter the organic phase or, if denatured, appear at the interphase . Chloroform treatment is used to remove the phenol, and alcohol precipitation concentrates the DNA while removing nucleotides, amino acids, and low - molecular- weight oligonucleotides and peptides. 随后用酚︰氯仿进行抽提，以去除残留的蛋白质，这时蛋白质将进入有机相，或者假如己变性的话将呈现在有机相与水相之间。氯仿处理是为了去除苯酚，而乙醇沉淀处理则可以浓缩DNA，同时去除核苷酸、氨基酸以及低分子量的寡核苷酸和肽。 As a further precaution, ethylene diamine tetraace-tic acid EDTA is include-ed in the buffers to chelate Mg2+. This will reduce deoxyribonuclease DNase activity because of the Mg2+ requirement of these enzymes. 为了进一步保护DNA样品的完整性，通常需要在缓冲液中添加乙二胺四乙酸 EDTA 以整合Mg2+，这样将会减少脱氧核糖核酸酶 DNase 的活性，因为该酶必须在有Mg2+存在时才会起作用。 The protocol described here result in DNA that is easily cut with restriction enzymes and, therefore, is useful for Southern Hybridization studies. If the DNA is to be used to construct a clone bank, higher molecular weight DNA is desirable, so the vortex step should be eliminated. 本实验方法分离得到的染色体DNA样品易被限制酶切割，因此，较适用于Southern杂交的研究。如果所制备的基因组DNA是用于构建克隆文库，要求得到较大分子量的DNA，则必须省略其中的振荡步骤。 三、试剂与器材（Reagents and apparatus） . Instruments High speed refrigerated centrifuge（高速冷冻离心机）、 Glass homogenizer （玻璃匀浆器） 、 Electrophoresis System （电泳系统） 、 Ultra