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class="pro_detail_btn"> <a href="#order" class="prodtl_btn1">產品咨詢</a><a href="/contact.html" target="_blank" class="prodtl_btn2">聯系我們</a> </div> </div> <div id="x4vfcpe" class="clear"></div> </div>  <div class="x4vfcpe" id="ny_pro_box02"> <div class="x4vfcpe" id="con"> <ul id="tags"> <li id="x4vfcpe" class=selectTag><A onmouseover="selectTag('tagContent0',this)" href="javascript:void(0)"onFocus="this.blur()">產品介紹：</A> </li> </ul> <div class="x4vfcpe" id=tagContent> <div id="x4vfcpe" class="tagContent selectTag" id=tagContent0><p>酶切鑒定</p><p> 限制性內切酶是一種工具酶，這類酶的特點是具有能夠識別雙鏈DNA 分子上的特異核苷酸順序的能力，能在這個特異性核苷酸序列內，切斷DNA 的雙鏈，形成一定長度和順序DNA 片段。對環(huán)狀質粒DNA有多少切口，就能產生多少酶切片段，因此鑒定酶切后的片段在電泳凝膠的區(qū)帶數，就可以推斷酶切口的數目，用已知分子量的線狀DNA 為對照，從片段的遷移率可以大致判斷酶切片段大小的差別。</p><p>酶切鑒定操作步驟<br />1．培養(yǎng)細菌<br />將帶有質粒的大腸桿菌DH5α接種在LB 瓊脂培養(yǎng)基上，37℃培養(yǎng)24~48h。<br />2．從細菌中快速提取制備質粒DNA。</p><p>3．質粒DNA 的酶解<br />將自提質粒加入20μL 的 TE 緩沖液，使 DNA *溶解。取清潔、干燥、滅菌的離心管編號用微量加樣器按各種試劑分別加入每個離心管內。<br />4.DNA酶切加樣要求：</p><p> 1）.選擇合適的酶切位點，并找到*緩沖液的酶切Buffer，可保證幾乎100%的酶活性.</p><p> 2）.每小時10-15U/ul的酶可以酶切1ug的質粒DNA,按照比例計算酶的使用量，酶量的加倍，可適當減少酶切時間。</p><p> 3）.質粒DNA的量通常在反應體系中擴大10倍量，確保酶切*。1小時內，50μl反應體積中，降解1μg的底物DNA所需的酶為一個活力單位。因此酶：DNA的反應比例可以由此確定。較小的反應體積更容易受到移液器誤差的影響。為了將甘油的濃度控制在5%以下，要注意酶的體積不要超過總體積的10%（一般酶都貯存于50%的甘油中）。 </p><p> 4）.有的酶要求100μg/ml的BSA以實現*活性。在這種情況下，我們也相應提供100X的BSA（10mg/ml）。不需要BSA的酶如果加了BSA也不會受太大影響。 </p><p> 5）.補無菌雙蒸水至相應體系，依實際情況做相應調整，加樣后，小心混勻，置于37℃水浴中，酶解2~3h，反應終止后，各酶切樣品于冰箱中貯存?zhèn)溆谩?br />5．DNA 瓊脂糖凝膠電泳<br /> （1）瓊脂糖凝膠的制備：稱取0.6g 瓊脂糖，置于三角瓶中，加入50 mL TBE 緩沖液，經微波爐加熱全部融化后，取出搖勻，此為1.2%的瓊脂糖凝膠。<br /> （2）膠板的制備：將冷卻至65℃左右的瓊脂糖凝膠液，小心倒入凝膠液，使膠液緩慢展開，直到在整個玻璃板表面形成均勻的膠層，室溫下靜置30 min，待凝固*后，輕輕拔出樣品槽模板，在膠板上即形成相互隔開的樣品槽。<br /> （3）加樣：用微量加樣器將上述樣品分別加入膠板的樣品小槽內。每次加完一個樣品，要用蒸餾水反復洗凈微量加樣器，以防止相互污染。<br />6．電泳<br />加完樣品后的凝膠板，立即通電。樣品進膠前，應使電流控制在20mA，樣品進膠后電壓控制在60~80V，電流為40~50 mA。當指示前沿移動至距離膠板1~2cm 處，停止電泳。<br />7、結果與觀察<br />在波長為254nm 的紫外燈下，觀察DNA 存在處顯示出的熒光條帶。<br /> </p></div> <div class="x4vfcpe" id="nr_textbox"><link rel="stylesheet" type="text/css" 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