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CopyControl™ cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells
CopyControl™ cDNA, Gene & PCR Cloning Kit w/ Chem. Comp Cells
货号
CCPCR1CC
规格
20 Rxns
存储温度
Chemically Competent Cells are stored at -70℃, the remainder is stored at -20℃
产品详情
Applications
- 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.
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.
Benefits
- 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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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. |
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| Table 1. PCR product cloning results with the CopyControl™ System. | |||||||||||||||||
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Figure 2. Single-copy CopyControl™ clones can be induced to 200 copies per cell. DNA from an equal number of uninduced (–) cells (lane 1) and undiluted induced (+) cells (lane 5) containing a 10-kb CopyControl PCR clone was analyzed by electrophoresis in a 1% agarose gel. Copy number in induced cells was estimated by visually comparing the staining intensity of the diluted induced sample with the uninduced sample. DNA from induced cells was diluted 1:200 (lane 2), 1:100 (lane 3), and 1:50 (lane 4). |
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Figure 3. Screening of clones from a 5-kb PCR product. The PCR product was cloned using the CopyControl™ cDNA, Gene, and PCR Cloning Kit. Very small portions from nine white colonies were screened using the kit, confirming the presence of the 5-kb insert. Lane M, DNA size ladder; lane 1, 8-kb supercoiled DNA marker; lanes 2-10, randomly chosen clones. Total time for colony screening, including gel electrophoresis, was approximately 60 minutes. |
*Covered by issued and/or pending patents.
| Catalog No. | Size |
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| CopyControl™ cDNA, Gene and PCR Cloning Kit (with electrocompetent cells) | |
| CCECPCR1 | 20 Reactions |
| Contents: 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. | |
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| CopyControl™ cDNA, Gene and PCR Cloning Kit (with chemically competent cells) | |
| CCPCR1CC | 20 Reactions |
| Contents: 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. | |
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| CopyControl™ Induction Solution | |
| CCIS125 | 25 ml |
| 1,000X concentrated solution. Filter sterilized. | |
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