EpiFOS™ Fosmid Library

Forward Sequencing Primer

Reverse Sequencing Primer

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

The EpiFOS™ Fosmid Library Production Kit provides all reagents needed to construct up to 10 complete and unbiased primary fosmid libraries using a novel cloning strategy (figure.1).

Genomic DNA is first sheared by passing it through a syringe needle (not supplied with the kit). Shearing the DNA into approximately 40-kb fragments leads to the highly random generation of DNA fragments in contrast to more biased libraries that result from fragmenting the DNA by partial restriction endonuclease digestion. The sheared DNA is end-repaired to generate 5'-phosphorylated blunt ends and size-selected using a low melting point agarose gel. Finally, the size-selected DNA is ligated into the supplied linearized and dephosphorylated pEpiFOS™-5 Fosmid Vector (Figure.2), packaged using ultra-high efficiency MaxPlax™ Lambda Packaging Extracts (>10⁹ pfu/µg for phage lambda), and plated on phage T1-resistant EPI100™-T1^R E. coli plating cells, all included in the kit. Fosmid pEpiFOS-5 has an F-factor origin, for replication in E. coli at single-copy, and cos sites that enable packaging of cosmid-sized clones (35-45 kb) in E. coli phage lambda packaging extracts. Propagation of fosmid clones at single copy results in more clones and improved clone stability compared to cloning in high-copy cosmids. The end result is a complete, unbiased and stable primary fosmid library.

Primers for end-sequencing pEpiFOS-5 clones are available separately.

Note: Fosmid clones constructed in the pEpiFOS-5 Vector cannot be induced to high-copy number. To construct a library of fosmid clones that can be induced to high-copy number, see the CopyControl™ Fosmid Library Production Kit.

Figure 1. Production of a fosmid library using the EpiFOS™ Fosmid Library Production Kit.

Figure 2. EpiFOS™-5 Fosmid Vector.

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

Protocols for: EpiFOS™ Fosmid Library Production

(catalogue number FOS0901)

(catalogue number F5FP010 / F5RP011)

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

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

References

1. Kim, U.J. et al. (1992) Nucl. Acids Res. 20, 1083.
2. Birren, B. et al. (1999) Construction of Bacterial Genomic Libraries in Genome Analysis: A Laboratory Manual v. 3, 241.

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