1质粒的提取及酶切.ppt

质粒DNA的提取及酶切 一、实验目的(Experimental Purpose) After the experiment is finished, students should be able to isolate plasmid DNA by the alkaline-detergent method. 掌握碱去垢剂法提取质粒DNA的方法。 Plasmids are small circular DNA molecules which replicate independently of the host genome and encode antibiotic resistance. They are the commonest vectors for carrying cloned DNA. Some small plasmids use the host’s enzymes to replicate. Larger plasmid may carry genes that encode their own replicative enzymes. Plasmids may be stringent ( low copy number ) or relaxed ( high copy number ) . 质粒分为：严紧型(低拷贝数)和松弛型(高拷贝数) Plasmids are frequently used as cloning vectors and alkaline lysis is one of the most widely used methods for the prepar-ation of plasmid DNA . 质粒通常用作克隆载体，而碱裂解法是制备质粒DNA最为常用的方法之一。 二、实验原理(Experimental Principle) 本方法是依据共价闭合环状质粒 DNA 与染色体 DNA 在变性和复性之间存在差异进行的。 When suspended in a solution of NaOH and sodium dodecyl sulfate (SDS), the cells are lysed by the high (alkaline) pH. Additionally, the protein and chromosome DNA will be denatured, and precipitate together with cell debris. In the presence of an acid solution (potassium acetate) and then centrifuged, the plasmid DNA were kept in the supernatant . After the addition of an alkaline solution, the plasmid DNA denaturation also occurred, but the close proximity of the two chains remain. When added to the acidic solution, each of the two chains of plasmid annealed with its complementary strand, thereby forming the original state. 三、试剂与器材（Reagents and apparatus） Ⅱ. Reagents 1. Solution Ⅰ（溶液Ⅰ ） ( 25mM Tris-HCl pH8.0, 50mM glucose, 10mM EDTA ) (25mM Tris·HCl（pH8.0）, 50mM 葡萄糖, 10mM EDTA) 2. Solution Ⅱ （溶液Ⅱ ） ( 0.4M NaOH, 2.0% SDS, made fresh just prior to use ) (0.4M NaOH,2% SDS [新鲜配制,等体积混和]) 3. Solution Ⅲ （溶液Ⅲ ） ( 5 M potassium acetate , acetic acid ) stored on ice . (5M KAC:60ml,冰醋酸:11.5ml,水:28.5ml) 4. TE buffer containing 10μg/ml RNase A ( preheated to 80℃ for 10 min inactivate DNase ) 5. 70% ethanol（乙醇）. 6.