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RNAi質(zhì)粒載體構(gòu)建服務(wù)|實驗技術(shù)服務(wù)

時間:2015/6/2閱讀:423
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關(guān)鍵詞:RNAi質(zhì)粒載體構(gòu)建服務(wù)|實驗技術(shù)服務(wù)
簡介:世界*品牌RNAi質(zhì)粒載體構(gòu)建服務(wù)|實驗技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
RNAi質(zhì)粒載體構(gòu)建服務(wù)|實驗技術(shù)服務(wù)——pCzn1質(zhì)粒+Arctic Express宿主菌低溫表達(dá)體系。
我們具備豐富的蛋白表達(dá)設(shè)計經(jīng)驗及實驗操作技巧,,承諾客戶不成功不收費。在獲得重組蛋白產(chǎn)品的同時,我們也會提供相應(yīng)的原始數(shù)據(jù)和原始圖片,不需要再額外花費精力重復(fù)實驗,。另一方面,我們所有的實驗數(shù)據(jù)真實可信,,我們提供原始的表達(dá)菌株和克隆質(zhì)粒,,客戶可以依據(jù)我們的實驗報告重復(fù)我們的實驗結(jié)果。
產(chǎn)品品牌:   http://www.,。,。com/goodsid/fenleiyi/2868603/1.html    
測序?qū)嶒灹鞒蹋?br>近年來的研究表明,一些短片斷的雙鏈RNA可以通過促使特定基因的mRNA降解來、特異的阻斷體內(nèi)特定基因表達(dá),誘使細(xì)胞表現(xiàn)出特定基因缺失的表型, 稱為RNA干擾(RNA interference,RNAi).siRNA(small interfering RNAs)就是這種短片斷雙鏈RNA分子,能夠以序列同源互補的mRNA為靶目標(biāo),降解特定的mRNA.RNAi的發(fā)現(xiàn)具有劃時代的意義,它不僅深入揭示 了細(xì)胞內(nèi)基因沉默的機制,而且它還是后基因組時代基因功能分析的有力工具,極大地促進(jìn)了人類揭示生命奧秘的進(jìn)程.現(xiàn)在越來越多的研究人員開始采用RNAi 來研究生物體的基因表達(dá).RNAi技術(shù)可廣泛應(yīng)用到包括功能基因組學(xué),藥物靶點篩選,細(xì)胞信號傳導(dǎo)通路分析,疾病治療等等.
目前為止較為常用的5種制備siRNAs的方法包括:
·化學(xué)合成
·體外轉(zhuǎn)錄
·長片斷dsRNAs經(jīng)RNase III 類降解 (e.g. Dicer, E. coli, RNase III)
·siRNA表達(dá)載體或者病毒載體在細(xì)胞中表達(dá)siRNAs
·PCR制備的siRNA表達(dá)框在細(xì)胞中表達(dá)
獲得高純度的siRNA產(chǎn)物是進(jìn)行實驗的*步,而轉(zhuǎn)染的效率則是非常關(guān)鍵的因素.
RNAi表達(dá)載體的構(gòu)建
1. 目的基因的確定
2,、設(shè)計siRNA靶序列
基本步驟:
(1)將100μl感受態(tài)細(xì)胞于冰上解凍.
(2)取5μl連接產(chǎn)物加入到感受態(tài)細(xì)胞中,輕輕旋轉(zhuǎn)幾次以 混勻內(nèi)容物. 在冰上放置30分鐘.
(3)將管放入預(yù)加溫到42℃的水浴中,熱激90秒.快速將管轉(zhuǎn)移到冰浴中,使細(xì)胞冷卻1~2分鐘.
(4)每管中加700μl LB培養(yǎng)基,37℃振蕩培養(yǎng)1小時,進(jìn)行復(fù)蘇.
(5)室溫4,000rpm離心5分鐘,棄去上清后,用剩余100μl培養(yǎng)基重懸細(xì)胞并涂布到含抗性的LB瓊脂平板表面.注意:細(xì)胞用量應(yīng)根據(jù)連接效率和感受態(tài)細(xì)胞的效率進(jìn)行調(diào)整.
(6)將平板置于室溫直至液體被吸收.
(7)倒置平皿,于37℃培養(yǎng),12~16小時后可出現(xiàn)菌落.

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