03哺乳動物組織基因組DNA提取

1、28.12.2020,1,Genomic DNA Extraction from Mammal Tissue,哺乳動物組織基因組DNA提取,28.12.2020,2,一、實(shí)驗(yàn)?zāi)康?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.掌握動物基因組DNA提取的操作方法。 2.掌握瓊脂糖凝膠電泳的操作的方法。,28.12.2020,3,二、

2、實(shí)驗(yàn)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.,本實(shí)驗(yàn)利用去垢劑SDS(十二烷基磺酸鈉)是為了使

3、蛋白變性并溶解細(xì)胞膜中的脂質(zhì)從而導(dǎo)致細(xì)胞裂解，而細(xì)胞裂解物又常用蛋白酶K和蛋白進(jìn)行水解處理。,28.12.2020,4,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

4、, and alcohol precipitation concentrates the DNA while removing nucleotides, amino acids, and low - molecular- weight oligonucleotides and peptides.,隨后用酚氯仿進(jìn)行抽提，以去除殘留的蛋白質(zhì)，這時(shí)蛋白質(zhì)將進(jìn)入有機(jī)相，或者假如己變性的話將呈現(xiàn)在有機(jī)相與水相之間。氯仿處理是為了去除苯酚，而乙醇沉淀處理則可以濃縮DNA，同時(shí)去除核苷酸、氨基酸以及低分子量的寡核苷酸和肽。,28.12.2020,5,As a further precaution, ethy

5、lene 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.,為了進(jìn)一步保護(hù)DNA樣品的完整性，通常需要在緩沖液中添加乙二胺四乙酸(EDTA)以整合Mg2+，這樣將會減少脫氧核糖核酸酶(DNase)的活性，因?yàn)樵撁副仨氃谟蠱g2+存在時(shí)才會起作用。,28.12.2020,6,The pro

6、tocol 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.,本實(shí)驗(yàn)方法分離得到的染色體DNA樣品易被限制酶切

7、割，因此，較適用于Southern雜交的研究。如果所制備的基因組DNA是用于構(gòu)建克隆文庫，要求得到較大分子量的DNA，則必須省略其中的振蕩步驟。,28.12.2020,7,三、試劑與器材（Reagents and apparatus）,. Instruments High speed refrigerated centrifuge（高速冷凍離心機(jī)）、 Glass homogenizer （玻璃勻漿器） 、 Electrophoresis System （電泳系統(tǒng)） 、 Ultraviolet transilluminator （紫外透射儀） 、 Ultra cold storage freez

8、er （超低溫冰箱） 、Autoclave （高壓滅菌鍋） 、 Pipettes （微量加樣器） 、Electronic balance （電子天平） 、 Acidometer （酸度計(jì)）,28.12.2020,8,. Material Liver of mice,28.12.2020,9,. Reagents,Tris、SDS、EDTA、 HCl、 NaOH 、 Sodium acetate、 Acetic acid、 Phenol、Chloroform、Ethanol、Isoamylol、 TE buffer 、 Protease K、RNase A、 Agarose 、DNA molec

9、ular weight markers 、Boric acid、Sugar、 Bromophenol blue、 Fluorescent dye(Gelview),28.12.2020,10,四、實(shí)驗(yàn)步驟(Experimental Procedures),冰浴處理生理鹽水 玻璃勻漿器 冰冷的生理鹽水清洗 配制工作液 處死小鼠 取出肝臟 組織勻漿液 酶解液 2ml勻漿液 組織細(xì)胞液 玻璃勻漿器勻漿 移至1.5ml離心管 5000rmin3060s 加400ul 吹散 加400ul 離心 無菌水 酶解液 水浴處理（55、1218h）,28.12.2020,11,四、實(shí)驗(yàn)步驟(Experimenta

10、l Procedures),等量分裝在兩支離心管內(nèi) 加入20ul的RNase 加入等體積提取液 4 10min 酶解樣品 待抽提的樣品 抽提 靜置 離心 配制 TE緩沖液 加等體積提取液500ul 10000rmin離心10min 4 10min 移出水相 至離心管 抽提 靜置 10000rmin離心10min 加1/10 加2倍 放入 離心 移出水相 NaAc 無水乙醇 -20 1-2h 12000rmin離心15min 加入 超低溫冰箱 融化、離心 棄上清液 洗滌 12000rmin離心15min 干燥 4 75%冷乙醇 離心 棄上清液 加TE 存放 DNA,28.12.2020,12,四

11、、實(shí)驗(yàn)步驟(Experimental Procedures),Agarose Gel Electrophoresis ： Preparation of the gel 制備0.3瓊脂糖凝膠 Loading DNA samples DNA samples 16l + Loading buffer4l Gel 電壓120V Gel photography,28.12.2020,13,Illuminate the gel with UV light and then take pictures. By comparing product bands with bands from the known molecular-weight markers, you should be able to identi