epicentre CCECPCR1 CopyControl™ cDNA, Gene & PCR Cloning Kit w

• 有效克隆zui多15 kb的任何插入片段，包括可能不稳定或编码对大肠杆菌  宿主细胞有毒的蛋白质的那些片段  。
• 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克隆存在于宿主细胞中，每个细胞仅约一个拷贝（P. Carninci和W. Szybalski，个人交流），则可以从库中获得多达25％的cDNA克隆，这可能是由于有害作用降低所致对宿主细胞的代谢和表达的克隆序列及基因产物的毒性较低。CopyControl™cDNA，基因和PCR克隆试剂盒*提供了一种快速且改进的方法（图1），用于克隆单拷贝数的任何平末端DNA片段（例如，基因组DNA，PCR产物或cDNA），并带有具有根据需要诱导高拷贝数的能力（图2）。试剂盒中提供的CopyControl pCC1™（Blunt）载体包含  大肠杆菌  F因子单拷贝复制起点（ori II）和诱导型高拷贝  oriV.使用ori V 不会发生复制，  除非并且直到在 TransforMax™EPI300™细胞的染色体（处于严格调控的启动子的控制下）中诱导出  trfA基因。因此，CopyControl cDNA，PCR或基因克隆可以单拷贝生长，以确保插入稳定性并成功克隆对宿主细胞有毒的序列。然后，每个细胞zui多可诱导200个拷贝，以用于所有下游应用，从而获得高产量的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.

优势说明

• 获得更多的cDNA或PCR克隆和更完整的克隆文库（表1）。
• Obtain more cDNA or PCR clones and more complete clone libraries (Table 1).
• 获得较少的突变克隆。以高拷贝数选择性地偏向对宿主细胞有毒性的基因中具有突变的克隆。
• Obtain fewer mutated clones. Clones having mutations in genes that are toxic to the host cell are selectively favored at high-copy number.
• 在1小时或更短的时间内筛选克隆的插入片段大小，而无需过夜培养或限制性内切酶消化（图3）。
• Screen clones for insert size in 1 hour or less without the need for overnight cultures or restriction endonuclease digests (Fig. 3).

图1（单击放大）。使用CopyControl™cDNA，基因和PCR克隆试剂盒，可在24小时内完成长达15 kb的插入片段的平末端平末端高效克隆。 为了提高克隆的稳定性，每个细胞可以将克隆保持一个拷贝，然后，只要需要，任何克隆都可以被诱导为高拷贝数（每个细胞zui多200个拷贝），从而获得高产量的DNA。  

Figure 1 (click to enlarge). Efficient, blunt-end cloning of inserts up to 15 kb and screening of clones can be completed in 24 hours using the CopyControl™ cDNA, Gene, and PCR Cloning Kit. Clones can be kept at one copy per cell for clone stability and then, whenever desired, any clone can be induced to high-copy number (up to 200 copies per cell) for high yields of DNA.  

图2.单拷贝CopyControl™克隆每个细胞可诱导200个拷贝。 在1％琼脂糖凝胶中通过电泳分析了来自相同数量的未诱导（-）细胞（泳道1）和未稀释的诱导（+）细胞（泳道5）的DNA，该DNA包含10-kb CopyControl PCR克隆。通过目测比较稀释的诱导样品和未诱导样品的染色强度来估计诱导细胞中的拷贝数。将来自诱导细胞的DNA以1：200（泳道2），1：100（泳道3）和1:50（泳道4）稀释。

图3.从5-kb PCR产物中筛选克隆。 使用CopyControl™cDNA，基因和PCR克隆试剂盒克隆PCR产物。使用试剂盒筛选了来自九个白色菌落的极小部分，确认了5kb插入片段的存在。M泳道，DNA大小阶梯；泳道1，8 kb超螺旋DNA标记; 2-10道，随机选择的克隆。菌落筛选（包括凝胶电泳）的总时间约为60分钟。