CopyControl™ Fosmid Library Production Kit

CopyControl™ HTP Fosmid Library Production Kit

pCC1™/pEpiFOS™ Forward

pCC1™/pEpiFOS™ Reverse

pCC2 Forward Sequencing Primer

pCC2 Reverse Sequencing Primer

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Last modified: 2010-02-03 20:36:36

The CopyControl™ Fosmid Library Production Kits provide an efficient and improved method for constructing a library of cosmid-sized (approximately 40kb) clones. The CopyControl Fosmid Library Production Kit contains the original pCC1FOS™ Vector, while the recently introduced CopyControl™ HTP Fosmid Library Production Kit includes modified pCC1FOS Vector, called pCC2FOS (Figure 1). Both of these CopyControl™ Vectors contain both the E. coli F-factor single-copy origin of replication and the inducible high-copy oriV. CopyControl™ Fosmid clones are grown at single copy to ensure insert stability and successful cloning of encoded and expressed toxic protein. The CopyControl™ Fosmid clones can then be induced up to 50 copies per cell.

The pCC2FOS Vector contains a primer cassette that optimizes end-sequencing results, especially in a high-throughput setting. This primer cassette, engineered in conjunction with Agencourt Bioscience Corporation, eliminates wasteful vector-derived sequencing reads by having the 3' terminus of the forward and reverse primers anneal 3 nucleotides from the cloning site. In addition, the 7-base sequence at the 3'-end of each primer was specifically designed to minimize mispriming to any contaminating E. coli DNA present after template purification (Figure 2).

TransforMax™ EPI300™ E. coli, required for induction of the CopyControl Fosmid clones up to 50 copies per cell, are included with the CopyControl™ Fosmid Library Production Kits.

Figure 1. CopyControl™ Vector map. The CopyControl™ pCC1FOS™ and pCC2FOS™ Vectors for CopyControl Fosmid library production are supplied linearized at the Eco72 I (blunt) site and then dephosphorylated and is ready for cloning end-repaired (blunt end) genomic DNA of approximately 40 kb.

The kit utilizes a strategy of cloning blunt-ended DNA fragments generated by random shearing of the DNA, to produce more complete and unbiased genomic libraries than can be obtained by partial restriction endonuclease digests. Genomic DNA is first sheared into approximately 40-kb fragments. The sheared DNA is end-repaired to generate blunt, 5'-phosphorylated ends and then size-selected by and recovered from a low melting-point agarose (LMP agarose) gel. Finally, the size-selected DNA is ligated into the cloning-ready CopyControl pCC1FOS or pCC2FOS Vector, packaged using ultra-high efficiency MaxPlax™ Lambda Packaging Extracts (>10⁹ pfu/µg DNA), included in the kit, and plated on the supplied TransforMax™ EPI300™ E. coli.

Figure 2. The CopyControl™ pCC2FOS™ Vector differs from the pCC1FOS™ Vector by the insertion of a new primer cassette thateliminates wasteful vector-derived sequencing reads and minimizes the potential for priming on the E. coli genome.

Figure 3. Typical sequencing results obtained with the pCC2FOS Forward Primer on a pCC2FOS clone at 1/48x BigDye dilution. Similar results were obtained with the pCC2FOS Reverse Primer (data not shown).

Figure 4. Overview of the process for preparing a fosmid library using CopyControl™ Fosmid Library Production Kits. Once the library has been prepared, individual clones can be cultured in small volume and induced to multiple-copy number for high yields of high purity DNA for fingerprinting, sequencing, etc.

Last modified: 2009-05-18 14:12:23

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Last modified: 2010-02-03 20:36:36

Protocols for: CopyControl™ Fosmid Library Production

(catalogue number CCFOS110)

(catalogue number CCPCR1CC)

(catalogue number HTFP061 / HTRP062)

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

Last modified: 2008-05-15 10:42:15

Customer References

CCFOS110 (1 Kit)

"Excellent product. High quality and high yielding results. Would recommend and buy it again. Highly satisfied"
-Dr Mahmut Tor Warwick HRI

Last modified: 2008-12-15 11:53:37