CopyControl? cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells

epicentre?CCPCR1CC?20 Rxns?CopyControl? cDNA

產(chǎn)品簡(jiǎn)介：北京中北林格供應(yīng)CopyControl? cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells，中北林格是epicentre總代理，提供epicentre產(chǎn)品貨號(hào)報(bào)價(jià)查詢。其他包括lucigen總代理，cellscript總代理，Biosearch technologies總代理，illumina總代理，ICLLAB總代理，ImmunoReagents總代理。

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產(chǎn)品名稱:

CopyControl? cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells

貨號(hào):CCPCR1CC

規(guī)格:20 Rxns

存儲(chǔ)溫度:

Chemically Competent Cells are stored at -70℃, the remainder is stored at -20℃

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CopyControl? cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells 詳細(xì)說(shuō)明

epicentre?CCPCR1CC?20 Rxns?CopyControl? cDNA?詳細(xì)說(shuō)明

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應(yīng)用領(lǐng)域

• 有效克隆最多15 kb的任何插入片段，包括可能不穩(wěn)定

  AIS107B

  或編碼對(duì)大腸桿菌宿主細(xì)胞有毒的蛋白質(zhì)的那些片段。
• Efficient cloning of any insert up to 15 kb, including those that may be unstable or encode proteins that are toxic to the?E. coli?host cells.

如果cDNA克隆存在于宿主細(xì)胞中，每個(gè)細(xì)胞僅約一個(gè)拷貝（P. Carninci和W. Szybalski，個(gè)人交流），則可以從庫(kù)中獲得多達(dá)25％的cDNA克隆，這可能是由于有害作用降低所致對(duì)宿主細(xì)胞的代謝和表達(dá)的克隆序列及基因產(chǎn)物的毒性較低。CopyControl?cDNA，基因和PCR克隆試劑盒*提供了一種快速且改進(jìn)的方法（圖1），用于克隆單拷貝數(shù)的任何平末端DNA片段（例如，基因組DNA，PCR產(chǎn)物或cDNA），并帶有具有根據(jù)需要誘導(dǎo)高拷貝數(shù)的能力（圖2）。試劑盒中提供的CopyControl pCC1?（Blunt）載體包含大腸桿菌?F因子單拷貝復(fù)制起點(diǎn)（ori?II）和誘導(dǎo)型高拷貝oriV.使用ori?V?不會(huì)發(fā)生復(fù)制，除非并且直到在TransforMax?EPI300?細(xì)胞的染色體（處于嚴(yán)格調(diào)控的啟動(dòng)子的控制下）中誘導(dǎo)出trfA基因。因此，CopyControl cDNA，PCR或基因克隆可以單拷貝生長(zhǎng)，以確保插入物穩(wěn)定性和對(duì)宿主細(xì)胞有毒序列的成功克隆。然后，每個(gè)細(xì)胞最多可誘導(dǎo)200個(gè)拷貝，以用于所有下游應(yīng)用，從而獲得高產(chǎn)量的DNA。

Up to ~25% more cDNA clones can be obtained in a library if the cDNA clones are present in the host cell at only about one copy per cell (P. Carninci and W. Szybalski, personal communication), presumably due to reduced detrimental effects on host cell metabolism and lower toxicity of expressed clone sequences and gene products. The CopyControl? cDNA, Gene, and PCR Cloning Kits* provide a rapid and improved method (Fig. 1) for cloning any blunt-end DNA fragments (e.g., genomic DNA, PCR products, or cDNA) at single-copy number, with the capability to induce to high-copy numbers as needed (Fig. 2). The CopyControl pCC1? (Blunt) Vector provided in the kits contains both the?E. coli?F-factor single-copy origin of replication (oriII) and an inducible high-copy?oriV. Replication does not occur using the?oriV unless and until the?trfA?gene in the chromosome of TransforMax? EPI300? cells (which is under the control of a tightly regulated promoter) is induced. Thus, CopyControl cDNA, PCR, or gene clones can be grown at single copy to ensure insert stability and successful cloning of sequences that are toxic to the host cell. Then, any desired clone can be induced up to 200 copies per cell for high yields of DNA for all downstream applications.

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好處

• 獲得更多的cDNA或PCR克隆和更完整的克隆文庫(kù)（表1）。
• 獲得較少的突變克隆。以高拷貝數(shù)選擇性地偏向?qū)λ拗骷?xì)胞有毒性的基因中具有突變的克隆。
• 在1小時(shí)或更短的時(shí)間內(nèi)篩選克隆的插入片段大小，而無(wú)需過(guò)夜培養(yǎng)或限制性內(nèi)切酶消化（圖3）。
• Obtain more cDNA or PCR clones and more complete clone libraries (Table 1).
• Obtain fewer mutated clones. Clones having mutations in genes that are toxic to the host cell are selectively favored at high-copy number.
• Screen clones for insert size in 1 hour or less without the need for overnight cultures or restriction endonuclease digests (Fig. 3).

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CopyControl? cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells?圖例說(shuō)明

epicentre?CCPCR1CC?20 Rxns?CopyControl? cDNA?圖例說(shuō)明

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圖1（單擊放大）。使用CopyControl?cDNA，基因和PCR克隆試劑盒，可在24小時(shí)內(nèi)完成長(zhǎng)達(dá)15 kb的插入片段的平末端平末端高效克隆。為了提高克隆的穩(wěn)定性，每個(gè)細(xì)胞可以將克隆保持一個(gè)拷貝，然后，只要需要，任何克隆都可以被誘導(dǎo)為高拷貝數(shù)（每個(gè)細(xì)胞最多200個(gè)拷貝），從而獲得高產(chǎn)量的DNA。

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圖2.單拷貝CopyControl?克隆每個(gè)細(xì)胞可誘導(dǎo)200個(gè)拷貝。在1％瓊脂糖凝膠中通過(guò)電泳分析了來(lái)自相同數(shù)量的未誘導(dǎo)（-）細(xì)胞（泳道1）和未稀釋的誘導(dǎo)（+）細(xì)胞（泳道5）的DNA，該DNA包含10-kb CopyControl PCR克隆。通過(guò)目測(cè)比較稀釋的誘導(dǎo)樣品和未誘導(dǎo)樣品的染色強(qiáng)度來(lái)估計(jì)誘導(dǎo)細(xì)胞中的拷貝數(shù)。將來(lái)自誘導(dǎo)細(xì)胞的DNA以1：200（泳道2），1：100（泳道3）和1:50（泳道4）稀釋。

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圖3.從5-kb PCR產(chǎn)物中篩選克隆。使用CopyControl?cDNA，基因和PCR克隆試劑盒克隆PCR產(chǎn)物。使用試劑盒篩選了來(lái)自九個(gè)白色菌落的極小部分，確認(rèn)了5kb插入片段的存在。M泳道，DNA大小階梯；泳道1，8 kb超螺旋DNA標(biāo)記;?2-10道，隨機(jī)選擇的克隆。菌落篩選（包括凝膠電泳）的總時(shí)間約為60分鐘。

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貨號(hào)尺寸

CopyControl?cDNA，基因和PCR克隆試劑盒（帶電感受態(tài)細(xì)胞）???CCECPCR120個(gè)反應(yīng)內(nèi)容：?CopyControl?pCC1?（平直克隆就緒）載體，PCR沉淀溶液，PCR 10X克隆緩沖液，末端修復(fù)酶混合物，F(xiàn)ast-Link?DNA連接酶，EpiLyse?解決方案，EpiBlue?解決方案，CopyControl?Fosmid自動(dòng)誘導(dǎo)解決方案，對(duì)照PCR產(chǎn)物，超螺旋DNA大小標(biāo)記，無(wú)菌水，具有電抗性的TransforMax?EPI300??大腸桿菌。

CopyControl?cDNA，基因和PCR克隆試劑盒（具有化學(xué)感受態(tài)細(xì)胞）???CCPCR1CC20個(gè)反應(yīng)內(nèi)容：?CopyControl?pCC1?（平直克隆就緒）載體，PCR沉淀溶液，PCR 10X克隆緩沖液，末端修復(fù)酶混合物，F(xiàn)ast-Link?DNA連接酶，EpiLyse?解決方案，EpiBlue?解決方案，CopyControl?Fosmid自動(dòng)誘導(dǎo)解決方案，對(duì)照PCR產(chǎn)物，超螺旋DNA大小標(biāo)記，無(wú)菌水，具有化學(xué)活性的TransforMax?EPI300??大腸桿菌。

CopyControl?感應(yīng)解決方案???CCIS12525毫升1,000X濃縮溶液。過(guò)濾器已消毒。

Catalog No.Size

CopyControl? cDNA, Gene and PCR Cloning Kit (with electrocompetent cells)???CCECPCR120 ReactionsContents:?CopyControl? pCC1? (Blunt Cloning-Ready) Vector, PCR Precipitation Solution, PCR 10X Cloning Buffer, End-Repair Enzyme Mix, Fast-Link? DNA Ligase, EpiLyse? Solution, EpiBlue? Solution, CopyControl? Fosmid Autoinduction Solution, Control PCR Product, Supercoiled DNA Size Marker, Sterile Water, Electrocompetent TransforMax? EPI300??E. coli.

CopyControl? cDNA, Gene and PCR Cloning Kit (with chemically competent cells)???CCPCR1CC20 ReactionsContents:?CopyControl? pCC1? (Blunt Cloning-Ready) Vector, PCR Precipitation Solution, PCR 10X Cloning Buffer, End-Repair Enzyme Mix, Fast-Link? DNA Ligase, EpiLyse? Solution, EpiBlue? Solution, CopyControl? Fosmid Autoinduction Solution, Control PCR Product, Supercoiled DNA Size Marker, Sterile Water, Chemically Competent TransforMax? EPI300??E. coli.

CopyControl? Induction Solution???CCIS12525 ml1,000X concentrated solution. Filter sterilized.

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