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                                                                                           shRNA雙鏈引物合成

產品介紹

根據我們的經驗,脫鹽純化的寡核苷酸足以滿足連接和克隆需要,如果您連接和克隆遇到困難,可以考慮換用PAGE純化或HPLC純化的引物,高純度的DNA oligos有助于您實驗的成功。

訂購流程
1.質量保證 2.發貨時間 3.關于售后
Transheep 有一套成熟的質檢程序,產品發貨前均經過質量檢測,確保您收到的產品質量無誤! Transheep擁有大量的基因現貨產品,本公司的現貨產品從訂貨日期起一周以內即可到貨,大大縮短了您的等待時間!另外Transheep的一站式克隆平臺也能讓您體驗到前所未有的快速定制服務! Transheep所售出的產品全部登記在冊,如果您拿到我們的產品覺得和實質不相符或存在問題,您只需要告訴我們相關產品信息我們會迅速做出反應,為您提供解決方案或退款!
4.訂購流程 5.聯系我們 6.相關服務:
聯系我們→客戶或銷售人員與您溝通→簽訂合同→支付預付款→執行項目→確認收貨→支付尾款→售后服務 Transheep提供免費的技術咨詢,任何關于產品或者技術問題,都可以聯系我們們。 免費熱線:400-0179890 服務郵箱:[email protected] 基因合成
亞克隆構建
3,UTR驗證實驗
啟動子合成及驗證
慢病毒包裝
腺病毒包裝
穩轉株構建

實驗流程
概述:
以下為使用pRI載體的方法概述。
1、將合成的正義和反義寡核苷酸鏈退火。
2、用BglII和XhoI雙酶切載體。
3、將退火的寡核苷酸鏈與酶切后的線性載體連接。
4、連接產物轉化大腸桿菌。
5、轉染細胞。
6、觀測熒光表達(含有GFP的載體)或篩選穩定表達細胞(含有抗性的載體)。
7、檢測目標基因蛋白或mRNA表達水平
載體構建
對于實驗中的很多步驟,您可以使用您實驗室中常用的方法,或者您的實驗經驗證實有效的方法。對于酶切、DNA純化以及轉化等步驟,您可以參照《分子克隆實驗指南》進行,也可以參照您購買的試劑盒中生產商提供的使用說明進行。
步驟1:退火寡核苷酸鏈
用水將寡核苷酸稀釋為100 μM。按以下體系配制退火反應體系:
正義寡核苷酸(100 μM) 5μl
反義寡核苷酸(100 μM) 5μl
NaCl                 100 mM
Tris-Cl pH7.4            50mM
加水補足                50μl
將配制好的退火反應緩沖液重復混合,短暫離心后放置PCR以上,運行以下程序:90℃ 4min,70℃ 10min,55℃ 10min,40℃ 10min,25℃ 10min。退火后的寡核苷酸可以立刻使用或者在-20℃長期保存。
步驟2:酶切載體
用XhoI和BglII雙酶切2μg載體。酶切方法和體系參照您購買的內切酶說明書或者按照您的實驗室習慣的方法進行。
通常情況下用大約20-30單位的酶在大約3小時可以酶切完全。
酶切后我們建議用瓊脂糖凝膠回收線性化載體。將回收后的載體體積稀釋為30μl。          
步驟3:連接載體
用水將退火后寡核苷酸稀釋100倍備用。按照以下體系配制連接反應體系:
T4 DNA 連接酶     5U
線性化載體    2μl
稀釋后寡核苷酸    2μl
10×連接酶Buffer   1μl
加水補足          10μl
接連反應條件和時間參照您購買的連接酶說明書進行。
為了減少空載體自連,連接反應完成后,在連接反應體系中加入1μl BglII,37℃反應30min,切割連接上的空載體。(選做)
步驟4:轉化大腸桿菌感受態
用連接后產物轉化大腸桿菌感受態細胞。市場上常見的大腸桿菌感受態細胞(例如Top10,DH5α)均可以使用,您也可以按照分子克隆中的方法自己制備感受態細胞。轉化方法按照供應商的說明書或者您實驗室中常用的方法進行。
在氨芐抗性的瓊脂平板上37℃培養轉化后細菌,大約14-16小時后,平板上出現單個細菌菌落。挑取多個菌落至氨芐抗性的培養基中培養后進行鑒定。
鑒定方法可以采用PCR鑒定或酶切鑒定。
步驟5:轉染細胞
pRI系列載體可用常用的轉染方法進行細胞轉染。包括:磷酸鈣轉染、脂質體轉染、電穿孔轉染等。您可以購買市售的商品化轉染試劑并且參照供應商說明書進行細胞轉染實驗。
步驟6:觀測GFP熒光和穩定表達細胞系篩選
如果細胞轉染成功,并且您使用了帶GFP的載體,根據細胞生長速度的不同,在轉染后3-5天能在熒光顯微鏡下觀察到細胞發出綠色熒光。請注意,綠色熒光蛋白的表達表明轉染細胞成功,不能說明RNA干擾實驗成功。
如果您使用了帶有真核抗性標簽如Neo-R的載體,這時您可以加入合適濃度的相應藥物如G418進行穩定表達細胞株的篩選。
步驟7:檢測RNA干擾效率
您可以在蛋白水平或mRNA水平檢測RNA干擾效率。一般情況下蛋白的表達變化與mRNA水平的表達變化一致, 也有少數情況下mRNA表達水平變化不及蛋白表達下降明顯。
為檢測蛋白表達情況,您可以使用Western-Blot。
為檢測mRNA表達情況,您可以使用逆轉錄+Realtime PCR或Northern-Blot。
具體檢測方法請參照分子克隆使用指南或者您實驗室的常用實驗方法。

 
疑難問題
如果RNA干擾效果不好,您需要重新檢查實驗步驟,看看是否有錯誤發生。如果一切沒有問題,請考慮以下問題:
  合成的寡核苷酸中錯誤的核苷酸較多,導致載體中連接上的寡核苷酸與您實際設計的寡核苷算不一致。做細胞轉染前,最好對鑒定后的載體進行測序。如果測序沒有正確的克隆,您可以考慮換一種純化方式或者換一個供應商。
  轉染效率不高。要達到良好的RNA干擾效果,需要較高的轉染效率配合,您可以采用帶有GFP的載體對轉染效率進行監測,必要時進行轉染實驗,試驗不同的轉染試劑和轉染條件對轉染效率的影響。
  設計的干擾序列不合適。我們建議同時設計3條干擾序列同時進行試驗,以便挑選出干擾效果好的序列。
您實驗中可能遇到的其它問題可能與您使用的特定細胞株、目標基因以及特殊實驗方法有關。您可以查閱相關文獻找到解決方法,或者聯系我們的技術支持[email protected],我們的技術人員將竭誠為您服務。

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