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class="x4vfcpe" id="newscont"> <h1 class="newsname">淺談基因的亞克隆</h1> <li id="x4vfcpe" class="time">發(fā)布日期：2013-11-15 瀏覽次數(shù)：4190 </li> <ul class="dcont"> <p><span style="font-size:16px"><span style="font-family:宋體"> <a ><strong>亞克隆</strong></a>：細(xì)胞克隆中，對(duì)培養(yǎng)的細(xì)胞來(lái)說，從原有的克隆中，再篩選出具有某種特性的細(xì)胞進(jìn)行培養(yǎng)，就是subclone，分為細(xì)胞克隆和分子克隆。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">一、</span><a ><strong><span style="font-family:宋體">亞克隆</span></strong></a><span style="font-family:宋體"><a ><strong>實(shí)驗(yàn)?zāi)康?/strong></a></span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">1、掌握細(xì)菌基因組提取的方法和原理。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">2、掌握限制性核酸內(nèi)切酶處理基因組及其結(jié)果分析。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">3、掌握基因片段回收的方法。</span></span></p><p> </p><p><span style="font-size:16px"><span style="font-family:宋體">二、<a ><strong>亞克隆</strong></a><strong><a >實(shí)驗(yàn)原理</a></strong></span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 細(xì)菌基因組的提?。和ㄟ^堿法或者酶法裂解細(xì)菌之后，可以將胞內(nèi)的核酸和蛋白質(zhì)全釋放出來(lái)。DNA溶于1mol/L的NaCl中，不溶于0.14mol/L的NaCl，而RNA溶于0.14mol/L的NaCl不溶于1mol/L的NaCl中，通過溶液中鹽濃度的變化可以將DNA和RNA分開。氯仿－異戊醇法除去蛋白，2倍體積乙醇將DNA沉淀出來(lái)。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 限制性核酸內(nèi)切酶只作用于特定的位點(diǎn)，處理基因組后只會(huì)得到有限的片斷。通過單酶切或者多酶切后電泳檢驗(yàn)就可以得到與物種本身有關(guān)的特定的圖譜。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 電泳后選定目的基因的條帶，紫外燈下將含有目的基因的凝膠切下，使用凝膠回收試劑盒，將凝膠溶化后，通過吸附柱時(shí)，核酸片段就吸附在柱上，洗脫液洗脫后加入2倍體積乙醇，核酸沉淀，TE溶解沉淀，核酸片段得到回收。</span></span></p><p> </p><p><span style="font-size:16px"><span style="font-family:宋體">三、<strong>試劑與器材</strong></span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">1、試劑 E.coli JM109，牛肉膏，胰蛋白胨，NaCl，微量細(xì)菌基因組抽提試劑盒（上海生工），BamH I、Xho1 核酸內(nèi)切酶（Takara），NEB 標(biāo)準(zhǔn)分子量片段（1kb DNA Ladder），凝膠電泳回收試劑盒</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">2、器材高速臺(tái)式冷凍離心機(jī)，水平電泳儀，漩渦混合儀，紫外檢測(cè)儀，凝膠成像分析系統(tǒng)，微量移液器及吸頭。</span></span></p><p> </p><p><span style="font-size:16px"><span style="font-family:宋體">四、<strong>操作方法</strong></span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">1．使用LB培養(yǎng)基活化、培養(yǎng)菌體。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">2．離心收集菌體，根據(jù)試劑盒說明提取基因組。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">3．限制性酶處理基因組。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">4．E.coli JM109的基因組經(jīng)過限制性內(nèi)切酶處理后，使用瓊脂糖凝膠電泳檢測(cè)，凝膠成像系統(tǒng)將酶切圖譜采集下來(lái)并對(duì)其分析。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">5．紫外燈下將需要回收的核酸片段切下來(lái)，使用凝膠回收試劑盒回收DNA片段。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">6．電泳檢測(cè)回收的核酸片段。</span></span></p><p> </p><p><span style="font-size:16px"><span style="font-family:宋體">五、<strong>關(guān)鍵步驟與注意事項(xiàng)</strong></span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">1．菌體培養(yǎng)過程要嚴(yán)格無(wú)菌，染菌后提取得到的基因組會(huì)成多條帶，酶切的圖譜也會(huì)比較復(fù)雜。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">2．基因組提取過程中所使用的器材要嚴(yán)格滅菌，防止核酸酶降解基因組。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">3．基因組提取過程不可以劇烈振蕩，防止DNA斷鏈。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">4．在內(nèi)切酶zui適條件下充分處理基因組，得到正確的圖譜。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">5．瓊脂糖溶化要充分。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">6．切下凝膠時(shí)要注意防護(hù)紫外線，電泳結(jié)束之后盡快將膠條切下來(lái)，凝膠條要窄，脫尾部分不回收。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> </span></span></p> </ul> <div 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