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#f30;"></span></p><p><span>產(chǎn)品關(guān)鍵詞：</span>江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù),熒光定量PCR</p><p><span>***更新：</span>2020-08-19 00:17:18</p><p><span id="click" >瀏覽次數(shù)：0次</span></p><a rel="nofollow" class="button">聯(lián)系我們</a></div></div><div id="yhdfwmt" class="rightbox"><h3>公司基本資料信息</h3><p style="border:0;"><a rel="nofollow">上海朝瑞生物科技有限公司</a></p><p><span>聯(lián)系人：</span>仲先生</p><p><span>郵箱: </span>scienceladder@163.com</p><p><span>電話: </span>18621867065</p><p><span>傳真: </span>021_</p><p><span>網(wǎng)址: </span></p><p><span>手機(jī): </span>021-54133071</p><p><span>地址: </span>上海市松江區(qū)廣富林路697號昂立大廈2113室</p><p>[當(dāng)前離線] <a rel="nofollow"> [加為商友] </a><a rel="nofollow"> [發(fā)送信件] </a></p></div></div><div id="d8cja3t" class="page-proshow02"><p class="title">詳細(xì)說明</p><div id="wf3pl7u" class="box"><p><p> 混合液在加入逆轉(zhuǎn)錄酶MMLV 之前先70℃干浴3分鐘，取出后立即冰水浴至管內(nèi)外溫度一致，然后加逆轉(zhuǎn)錄酶0，江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù)，江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù).5μl，37℃水浴60分鐘。 </p> <p> 取出后立即95℃干浴3分鐘，得到逆轉(zhuǎn)錄終溶液即為cDNA溶液，保存于-80℃待用。管家基因(β-actin)實(shí)時定量PCR </p> <p> β-actin陽性模板的標(biāo)準(zhǔn)梯度制備陽性模板的濃度為1011，反應(yīng)前取3μl按10倍稀釋(加水27μl并充分混勻)為1010，江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù)，依次稀釋至109、108、107、106、105、104，以備用。 </p> <p> 反應(yīng)體系如下： </p> <p> 標(biāo)準(zhǔn)品反應(yīng)體系序號反應(yīng)物劑量 1 SYBR Green 1 染料 10μl 2 陽性模板上游引物F 0.5μl 3 陽性模板下游引物R 0.5μl 4 dNTP 0.5μl 5 Taq酶 1μl 6 陽性模板DNA 5μl 7 ddH2O 32.5μl 8 總體積 50μl 輕彈管底將溶液混合，6000rpm短暫離心。江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù)</p><div style="width:100%;text-align: center;"><img style="max-width: 640px; max-height: 320px;" alt="江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù),熒光定量PCR" src="http://tyunfile.71360.com/UploadFile/shchaorui/637116253047636719/652001.png"></div><p> 使用包含 3 種不同濃度的 ROX 染料的預(yù)混液對 TGF-β 進(jìn)行擴(kuò)增。顯示 (A) Ct 值和 (B) 標(biāo)準(zhǔn)差隨 ROX 染料濃度的變化。降低 ROX 染料濃度導(dǎo)致 Ct 出現(xiàn)得更早，但是會增大標(biāo)準(zhǔn)差。使用 Applied Biosystems 7500 實(shí)時熒光定量 PCR 系統(tǒng)執(zhí)行所有擴(kuò)增。 </p> <p> Ct 隨 PCR 效率發(fā)生變化。藍(lán)色標(biāo)準(zhǔn)曲線的效率為 **（斜率為 –3.3）。綠色標(biāo)準(zhǔn)曲線的效率為 78%（斜率為 –4）。與高效率條件相比（藍(lán)色），在低效率條件下（綠色），擴(kuò)增量 Y 使 Ct 出現(xiàn)得更早。較低擴(kuò)增量 (X) 時則情況相反，與高效率條件（藍(lán)色）相比，低效率條件（綠色）使 Ct 出現(xiàn)得更晚。北京基因突變熒光定量PCR技術(shù)服務(wù)</p><div style="width:100%;text-align: center;"><img style="max-width: 640px; max-height: 320px;" alt="江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù),熒光定量PCR" src="http://tyunfile.71360.com/UploadFile/shchaorui/637116253057744141/7327894.png"></div><p> 自從1983年K. Mullis發(fā)明聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase chain reaction，PCR)以來，PCR技術(shù)很快成為科研、臨床診斷的熱點(diǎn)技術(shù)。 </p> <p> 而實(shí)時熒光定量PCR技術(shù)，顧名思義，就是在PCR的基礎(chǔ)上加入熒光基團(tuán)，通過熒光信號的變化的實(shí)時監(jiān)測PCR的反應(yīng)過程，***通過對未知模板進(jìn)行定量分析的方法。 </p> <p> 目前，實(shí)時熒光定量 PCR 已成為不同樣品間進(jìn)行基因表達(dá)水平定量差異比較的**性方法。在過去十幾年中，該方法迅速流行，涉及科學(xué)的多個領(lǐng)域，包括農(nóng)業(yè)、環(huán)境、工業(yè)和醫(yī)學(xué)研究。 </p><p> 熒光定量PCR 實(shí)驗(yàn)步驟（1） </p> <p> 樣品RNA的抽提 </p> <p> <br /> </p> <p> ①取凍存已裂解的細(xì)胞，室溫放置5分鐘使其完全溶解。 </p> <p> <br /> </p> <p> ②每樣品加入1ml的TRIZOL試劑裂解，蓋緊管蓋。手動劇烈振蕩管體15秒后，15到30℃孵育2到3分鐘。4℃下12000rpm離心15分鐘。離心后混合液體將分為下層的紅色酚相，中間層以及無色水相上層。RNA全部被分配于水相中。水相上層的體積大約是勻漿時加入的TRIZOL試劑的60%。 </p> <p> <br /> </p> <p> ③RNA沉淀將水相上層轉(zhuǎn)移到一干凈無RNA酶的離心管中。加等體積異丙醇混合以沉淀其中的RNA，混勻后15到30℃孵育10分鐘后，于4℃下12000rpm 離心10分鐘。此時離心前不可見的RNA沉淀將在管底部和側(cè)壁上形成膠狀沉淀塊。 </p> <p> <br /> </p> <p> ④RNA清洗移去上清液，每1mlTRIZOL試劑裂解的樣品中加入至少1ml的75%乙醇(75%乙醇用DEPCH2O配制)，清洗RNA沉淀?；靹蚝?，4℃下7000rpm離心5分鐘。 </p> <p> <br /> </p> <p> ⑤RNA干燥小心吸去大部分乙醇溶液，使RNA沉淀在室溫空氣中干燥5-10分鐘。 </p> <p> <br /> </p> <p> ⑥溶解RNA沉淀溶解RNA時，先加入無RNA酶的水40μl用移液器反復(fù)吹打幾次，使其完全溶解，獲得的RNA溶液保存于-80℃待用。 </p><div style="width:100%;text-align: center;"><img style="max-width: 640px; max-height: 320px;" alt="江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù),熒光定量PCR" src="http://tyunfile.71360.com/UploadFile/shchaorui/637116253043593750/8100830.png"></div><p style="text-indent:25px">Rn 值是由 Applied Biosystems? FAM? 染料的熒光除以 ROX 染料的熒光而計算得出的比率。因此，假定 FAM 染料產(chǎn)生的熒光信號保持不變，ROX 染料量越少，產(chǎn)生的 Rn 值就越高。這將導(dǎo)致 Rn 基線上升，以及隨后 ΔRn 的減少和 Ct 值的變化。通過降低 ROX 染料水平而得到不同 Ct 值，沒有對反應(yīng)的真實(shí)靈敏度產(chǎn)生任何影響，卻有著意想不到的后果。如圖 4 所示，降低 ROX 染料的濃度可能會增加 Ct 值的標(biāo)準(zhǔn)差。標(biāo)準(zhǔn)差越大，分辨靶濃度之間的細(xì)微區(qū)別的可信度就越低（參加下文的精確度一節(jié)）。天津非編碼RNA 熒光定量PCR分析服務(wù)</p><p style="text-indent:25px">江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù)</p><p> 熒光定量PCR實(shí)驗(yàn)步驟： </p> <p> 取凍存已裂解的細(xì)胞，室溫放置5分鐘使其完全溶解。 </p> <p> 兩相分離每1ml的TRIZOL試劑裂解的樣品中加入0.2ml的lvfang，蓋緊管蓋。手動劇烈振蕩管體15秒后，15到30℃孵育2到3分鐘。4℃下12000rpm離心15分鐘。離心后混合液體將分為下層的紅色酚lvfang相，中間層以及無色水相上層。RNA全部被分配于水相中。水相上層的體積大約是勻漿時加入的TRIZOL試劑的60%。 </p> <p> RNA沉淀將水相上層轉(zhuǎn)移到一干凈無RNA酶的離心管中。加等體積異丙醇混合以沉淀其中的RNA，混勻后15到30℃孵育10分鐘后，于4℃下12000rpm 離心10分鐘。此時離心前不可見的RNA沉淀將在管底部和側(cè)壁上形成膠狀沉淀塊。江蘇轉(zhuǎn)基因熒光定量PCR技術(shù)服務(wù)</p><p style="text-indent:25px">上海朝瑞生物科技有限公司是一家1.發(fā)布科研技術(shù) 2.發(fā)布應(yīng)用技術(shù) 3.發(fā)布專業(yè)專長 4.發(fā)布科研成果 5.發(fā)布我的需求 6.發(fā)布篩選檢驗(yàn)檢測服務(wù) 7.發(fā)布協(xié)同代研發(fā)服務(wù) 8.發(fā)布升級改造產(chǎn)品服務(wù) 9.發(fā)布實(shí)驗(yàn)室及儀器設(shè)備共享 10.發(fā)布培訓(xùn)基地共享 11.發(fā)布工廠代加工及共享車間 12.發(fā)布科研/項(xiàng)目團(tuán)隊(duì)招聘13.發(fā)布研究生婚戀 14.發(fā)布項(xiàng)目整包服務(wù) 15.發(fā)布資源交換業(yè)務(wù)的公司，致力于成為客戶業(yè)務(wù)創(chuàng)新、化工可信賴的合作伙伴。公司自2003-01-22成立以來，投身于[ "科研技術(shù)服務(wù)", "應(yīng)用技術(shù)服務(wù)", "科研成果轉(zhuǎn)化", "科學(xué)產(chǎn)品銷售" ]，是化工的主力軍。公司堅(jiān)持以技術(shù)創(chuàng)新為發(fā)展引擎，以客戶滿意為動力，目前擁有5~10人專業(yè)人員，年?duì)I業(yè)額達(dá)到2000-3000萬元。上海朝瑞科技始終關(guān)注自身，在風(fēng)云變化的時代，我們對自身的建設(shè)毫不懈怠，高度的專注與執(zhí)著使我們在行業(yè)的從容而自信。</p></p><br /><p>文章來源地址: http://www.cdcfah.com/cp/961545.html</p></div></div></div></div></div><div id="nwrsyev" class="page-right twoColRt"><div id="r3vr7vg" class="box01 margin10 clearfix"><h3><a href="http://www.cdcfah.com/cp/">猜你喜歡</a></h3><div id="pjabh2w" class="recomPic likePro"><a href="http://www.cdcfah.com/cp/814588.html"><div><img src="http://tyunfile.71360.com/UpLoadFile/2019/11/1/14/637082168864691056_lgpj_5374157.jpg" alt="福建ZF配件哪家強(qiáng) 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