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class="about"> <div id="im8tiuhso" class="PageView"> <div class="im8tiuhso" id="nr_main_box">  <div id="im8tiuhso" class="pro_detail_main"> <div id="im8tiuhso" class="prodetail_img"> <div class="im8tiuhso" id="preview"> <div id="im8tiuhso" class="jqzoom" id="spec-n1"> <div class="im8tiuhso" id="ceshi" style=""> <img src="http://img51.chem17.com/gxhpic_33572a5ad1/6e95fd1ed072fae12efb5d9273dcd4eb16718c5634b3d89de5b7f37ecc5533e4a5ea0902bb05c767.jpg" jqimg="http://img51.chem17.com/gxhpic_33572a5ad1/6e95fd1ed072fae12efb5d9273dcd4eb16718c5634b3d89de5b7f37ecc5533e4a5ea0902bb05c767.jpg" /> </div> </div> <div class="im8tiuhso" id="spec-n5">  <div class="im8tiuhso" id="spec-list"> <ul class="list-h" > <li><p><img onclick="jQuery('.videoItem').hide();mybaidu.window.SetPause();" src="http://img51.chem17.com/gxhpic_33572a5ad1/6e95fd1ed072fae12efb5d9273dcd4eb16718c5634b3d89de5b7f37ecc5533e4a5ea0902bb05c767_800_800_5.jpg" alt="" data-zoom-image="http://img51.chem17.com/gxhpic_33572a5ad1/6e95fd1ed072fae12efb5d9273dcd4eb16718c5634b3d89de5b7f37ecc5533e4a5ea0902bb05c767_800_800_5.jpg" onload="DrawImage(this,460,300)"></p></li> </ul> </div>  </div> </div> </div> <div id="im8tiuhso" class="pro_detail_text"> <h1>目的基因亞克隆</h1> <div id="im8tiuhso" class="jyms"><p><span>簡(jiǎn)要描述：</span>對已經(jīng)獲得的目的DNA條帶進(jìn)行重新克隆，其目的在于對目的DNA進(jìn)行進(jìn)一步分析，或者進(jìn)行重組改造等。亞克隆的基本過(guò)程包括：（1）目的DNA條帶和載體的制備；（2）目的DNA條帶和載體的連接；（3）連接產(chǎn)物的轉化；（4）重組子篩選。</p></div> <ul> <li><img src="/Skins/270236/images/dot.png" /><span>產(chǎn)品型號：</span></li> <li><img src="/Skins/270236/images/dot.png" /><span>廠(chǎng)商性質(zhì)：</span>生產(chǎn)廠(chǎng)家</li> <li><img src="/Skins/270236/images/dot.png" /><span>更新時(shí)間：</span>2024-05-13</li> <li><img src="/Skins/270236/images/dot.png" /><span>訪(fǎng) 問(wèn) 量：</span>2762</li> </ul> <div id="im8tiuhso" class="pro_detail_btn"> <a href="#order" class="prodtl_btn1">產(chǎn)品咨詢(xún)</a><a href="/contact.html" target="_blank" class="prodtl_btn2">聯(lián)系我們</a> </div> </div> <div id="im8tiuhso" class="clear"></div> </div>  <div class="im8tiuhso" id="ny_pro_box02"> <div class="im8tiuhso" id="con"> <ul id="tags"> <li id="im8tiuhso" class=selectTag><A onmouseover="selectTag('tagContent0',this)" href="javascript:void(0)"onFocus="this.blur()">產(chǎn)品介紹：</A> </li> </ul> <div class="im8tiuhso" id=tagContent> <div id="im8tiuhso" class="tagContent selectTag" id=tagContent0><p><strong>亞克隆</strong></p><p>已經(jīng)獲得的目的DNA條帶進(jìn)行重新克隆，其目的在于對目的DNA進(jìn)行進(jìn)一步分析，或者進(jìn)行重組改造等。</p><p><strong>亞克隆的基本過(guò)程包括：</strong></p><p>（1）目的DNA條帶和載體的制備；（2）目的DNA條帶和載體的連接；（3）連接產(chǎn)物的轉化；（4）重組子篩選。</p><p><strong>目的DNA條帶和載體的制備</strong><br /> 選擇適宜的限制性核酸內切酶，消化已知目的DNA和載體，獲得線(xiàn)性DNA，用于重組。根據目的DNA和載體的具體情況，選擇一種或者兩種適當的限制酶切割，分別產(chǎn)生對稱(chēng)性粘性末端（用一種限制性?xún)惹忻高M(jìn)行消化而產(chǎn)生帶有互補突出端）、不對稱(chēng)粘性末端（用兩種不同的限制性?xún)惹忻高M(jìn)行消化而產(chǎn)生帶有非互補突出端）、平端。在亞克隆時(shí)，*不對稱(chēng)相容末端連接，次選對稱(chēng)性粘性相容性末端連接，由于平末端連接效率較低，通常很少采用。但有時(shí)目的片段的末端與載體不匹配，一般先將不匹配末端補平，然后再以平末端連接。</p><p><strong>利用T4 DNA連接酶進(jìn)行目的DNA條帶和載體的體外連接</strong></p><table><tbody><tr><td><p>外源DNA條帶末端性質(zhì)</p></td><td><p>連接要求</p></td><td><p> 連接結果</p></td></tr><tr><td><p>不對稱(chēng)粘性末端</p></td><td><p>兩種限制酶消化后，需純化載體以提高連接效率</p></td><td><p>載體與外源DNA連接處的限制酶切位點(diǎn)?？杀Ａ?；非重組克隆的背景較低；外源DNA可以定向插入到載體中。</p></td></tr><tr><td><p>對稱(chēng)性粘性末端</p></td><td><p>線(xiàn)形載體DNA常需磷酸酶脫磷處理</p></td><td><p>載體與外源DNA連接處的限制酶切位點(diǎn)?？杀Ａ?；重組質(zhì)粒會(huì )帶有外源DNA的串聯(lián)拷貝；外源DNA會(huì )以?xún)蓚€(gè)方向插入到載體中。</p></td></tr><tr><td><p>平端</p></td><td><p>要求高濃度的DNA和連接酶</p></td><td><p>載體與外源DNA連接處的限制酶切位點(diǎn)消失；重組質(zhì)粒會(huì )帶有外源DNA的串聯(lián)拷貝；非重組克隆的背景較高。</p></td></tr></tbody></table><p><strong>連接產(chǎn)物的轉化</strong><br />⑴ 取100μl貯存于-70℃鈣化菌，冰浴化開(kāi)；<br />⑵ 加入適量連接產(chǎn)物（一般不超過(guò)10μl，輕輕混勻，冰浴20min；<br />⑶ 于42℃熱休克90s，迅速轉移至冰浴中，繼續冰浴2-3min；<br />⑷ 加入LB液體培養基200μl，于37℃緩搖孵育45min；<br />⑸ 將培養物適量涂于1.5%瓊脂LB平板（根據質(zhì)粒性質(zhì)添加抗生素或/和X-Gal/IPTG），待膠表面沒(méi)有液體流動(dòng)時(shí)，37℃溫箱倒置培養12-16hr。</p><p><strong>重組子的篩選</strong><br /> 根據載體的遺傳特征篩選重組子，如α-互補、抗生素基因等?，F在使用的許多載體都帶有一個(gè)大腸桿菌的DNA的短區段，其中有β-半乳糖苷酶基因（lacZ）的調控序列和前146個(gè)氨基酸的編碼信息。在這個(gè)編碼區中插入了一個(gè)多克隆位點(diǎn)（MCS），它并不破壞讀框，但可使少數幾個(gè)氨基酸插入到β-半乳糖苷酶的氨基端而不影響功能，這種載體適用于可編碼β-半乳糖苷酶C端部分序列的宿主細胞。因此，宿主和質(zhì)粒編碼的片段雖都沒(méi)有酶活性，但它們同時(shí)存在時(shí)，可形成具有酶學(xué)活性的蛋白質(zhì)。這樣，lacZ基因在缺少近操縱基因區段的宿主細胞與帶有完整近操縱基因區段的質(zhì)粒之間實(shí)現了互補，稱(chēng)為α-互補。由α-互補而產(chǎn)生的LacZ 細菌在誘導劑IPTG的作用下，在生色底物X-Gal存在時(shí)產(chǎn)生藍色菌落，因而易于識別。然而，當外源DNA插入到質(zhì)粒的多克隆位點(diǎn)后，幾乎不可避免地導致無(wú)α-互補能力的氨基端片段，使得帶有重組質(zhì)粒的細菌形成白色菌落。這種重組子的篩選，又稱(chēng)為藍白斑篩選。如用藍白斑篩選則經(jīng)連接產(chǎn)物轉化的鈣化菌平板37℃溫箱倒置培養12-16hr后，有重組質(zhì)粒的細菌形成白色菌落。</p><p> </p><p> </p><p> </p></div> <div class="im8tiuhso" id="nr_textbox"><link rel="stylesheet" type="text/css" href="/css/MessageBoard_style.css"> <a name="order" id="order"></a> <div id="im8tiuhso" class="ly_msg" id="ly_msg"> <form method="post" name="form2" id="form2"> <h3>留言框 </h3> <ul> <li> <h4 class="xh">產(chǎn)品：</h4> <div id="im8tiuhso" class="msg_ipt1"><input class="textborder" size="30" name="Product" id="Product" value="目的基因亞克隆" placeholder="請輸入產(chǎn)品名稱(chēng)" /></div> </li> <li> <h4>您的單位：</h4> <div id="im8tiuhso" class="msg_ipt12"><input class="textborder" size="42" name="department" id="department" placeholder="請輸入您的單位名稱(chēng)" /></div> </li> <li> <h4 class="xh">您的姓名：</h4> <div id="im8tiuhso" class="msg_ipt1"><input class="textborder" 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