CopyControl™ cDNA, Gene & PCR Cloning Kit (chem competent cells)

CopyControl™ cDNA, Gene & PCR Cloning Kit (electro cells)

CopyControl™ Induction Solution

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Last modified: 2008-07-15 14:57:15

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 (personal communication from P. Carninci and W. Szybalski labs), presumably due to reduced detrimental effects on host cell metabolism and lower toxicity of expressed clone sequences and gene products. CopyControl™ cDNA, Gene & PCR Cloning Kits provide a rapid and improved method for cloning any blunt-end DNA fragments, such as blunt-end genes, PCR products, or cDNAs, at single-copy, with the capability to induce to high-copies at will.

The CopyControl™ pCC1™ (Blunt) Vector provided in the kits contains both the E. coli F-factor single-copy origin of replication (ori) 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 at will to up to 200 copies per cell for high yields of DNA for all downstream applications.

Figure 1. Efficient, blunt-end cloning of any cDNA, PCR product or other dsDNA fragment up to 15 kb and screening of clones can be completed in 24 hours using the CopyControl™ cDNA, Gene & 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.

Table 1. PCR Product Cloning Results Size of the PCR Product Cloned cfu/µg of PCR Product Percent full-Length Clones 1.4kb >1 x 107 >90% 5kb 1 x 107 90% 10kb 3.5 x 106 80% 15kb 9.2 x 105 30%

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 electrophoresed 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).

Last modified: 2008-05-14 16:46:46

Benefits

• Get more cDNA or PCR clones and more complete clone libraries by efficiently cloning any PCR product or cDNA up to 15 kb, including those that may be unstable or encode proteins that are toxic to the E. coli host cells
• Obtain fewer mutated clones. Clones having mutations in genes that are toxic to the host cell are selectively favored at high-copy
• Keep clones or a clone library at one copy per cell for insert stability. Then, induce up to 200 copies per cell at will
• Screen the size of the clones in 1 hour or less without the need for overnight cultures or restriction endonuclease digests.

Last modified: 2008-05-14 16:46:46

Protocols for: CopyControl™ cDNA, Gene & PCR Cloning Kits (BamH, EcoR I, Hind III)

(catalogue number CCECPCR1)

(catalogue number CCPCR1CC)

(catalogue number CCIS125)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46