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1、2023/6/25,1,质粒DNA的提取及酶切,Isolation of Plasmid DNA and Enzymatic Digestion,2023/6/25,2,一、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。,2023/6/25,3,Plasmids are small circular DNA molecules
2、 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 hosts enzymes to replicate.Larger plasmid may carry genes that encode their own replicative enzymes.,质粒是存在于几乎所有细菌中染色体之外的外状DNA分子。质粒通常携带
3、有染色体上所不存在的基因，并表现出一些有用的性状，如抗生素抗性，耐受重金属等。质粒拥有自己的复制原点，因此可以不依赖于染色体而进行独立复制。一些小的质粒利用宿主细胞的酶进行复制，而较大的质粒则自身携有编码与复制有关的酶。,2023/6/25,5,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
4、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。,2023/6/25,6,二、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.Add
5、itionally,the protein and chromosome DNA will be denatured,and precipitate together with cell debris.,This method is based on the different rates of denaturation and renaturation of covalently closed circular plasmid DNA and chromosomal DNA.,当细胞悬浮于 NaOH 和十二烷基酸钠(SDS)溶液中时，在高pH(碱)的作用下细胞发生裂解，此外，蛋白质和染色体D
6、NA发生变性与细胞碎片一起沉淀下来。,2023/6/25,7,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
7、 the acidic solution,each of the two chains of plasmid annealed with its complementary strand,thereby forming the original state.,加入中和溶液(乙酸钾)溶液后离心，则质粒DNA则留在上清液中。,当加入碱溶液时质粒DNA也发生变性，但其两条链仍然靠得很近，就像一条链上的两个环链，当加入酸性溶液进行中和时，质粒DNA的两条链就分别与其互补链重新退火，进而形成原始状态的质粒。,2023/6/25,8,三、试剂与器材（Reagents and apparatus）,.Ins
8、truments1.Constant temperature incubator（恒温培养箱）2.Constant temperature shaking table（恒温摇床）3.High speed centrifuge（高速离心机）4.Vortex mixer（涡旋振荡器）5.Clean Benches（超净工作台）6.Autoclave（高压灭菌锅）7.Pipettes（微量加样器）,2023/6/25,9,.Reagents,1.Solution（溶液）(25mM Tris-HCl pH8.0,50mM glucose,10mM EDTA)(25mM TrisHCl（pH8.0）,5
9、0mM 葡萄糖,10mM EDTA)2.Solution（溶液）(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),2023/6/25,10,3.Solution（溶液）(5 M potassium acetate,acetic acid)stored on ice.(5M KAC:60ml,冰醋酸:11.5ml,水:28.5ml)4.TE buffer containing 10g/ml RNase A(preheated to 80 for 10 min inactivate DNase)
10、5.70%ethanol（乙醇）.6.Phenol:chloroform 平衡酚:氯仿(1:1),2023/6/25,11,7.LB培养基：,胰化蛋白胨 10g 酵母提取物 5g NaCl 10g pH 7.0 摇动容器直至溶质完全溶解，用5mol/L氢氧化钠（约0.2ml）调节pH值至7.0，加入去离子水至总体积为1000ml，在1.034105Pa高压蒸汽灭菌20min。8.菌种：含质粒的大肠杆菌,2023/6/25,12,.Preparation of Plasmid DNA取种子液4-6 ml于含有50g/ml卡那霉素的100 ml LB培养基中 37振荡培养（16 h）至OD600=
11、1.0收集1.4 ml菌体于1.5 ml离心管中 4 4000 rpm 离心 2min 弃培养液，尽可能干燥将沉淀悬浮于100l冰冷的溶液，剧烈振荡 室温10 min,四、实验步骤(Experimental Procedures),2023/6/25,13,加入200l溶液（新鲜配置），颠倒混 冰浴5min加入200l冰冷的溶液，温和振荡 冰浴 15 min 4 12000 rpm 离心 15 min 上清液转移到另一离心管中（记录体积），往上清液中加入等体积酚：氯仿（1：1）反复振荡混匀4 12000 rpm 离心 5 min,2023/6/25,14,上清液转移到另一离心管中（记录体积），
12、往向上清液加入倍体积无水乙醇，振荡混匀，于室温静置5min 4，12000r/min 离心5min弃上清液，沉淀用 1ml 冰冷的70%乙醇洗涤 4，12000 rpm离心2min 弃上清液，挥发尽乙醇加入50l TE缓冲液溶解质粒DNA，-20冻存 第二次实验（酶切）,2023/6/25,15,.Enzymatic Digestion,取5l DNA溶液 Take 5l of sample of DNA.加入1l 酶切缓冲液和Eco RI酶1l（2U)Add 1l of Enzymatic Digestion buffer and 1l（2U)of Eco RI.补无菌水3l Add 3l of sterile water.37保温3h。Incubate at 37 for 3 hours.,2023/6/25,16,五、思考题(Questions),1.简要叙述溶液、溶液和溶液的作用，以及实验中分别加入上述溶液后，反应体系出现的现象及其成因。2.简要叙述酚氯仿抽提体系后出现的现象及其成因。3.沉淀时为什么要用无水乙醇及在高盐、低温条件下进行？,
