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VMRC-28: Stickleback fish (Gasterostetus aculealus) BAC Library |
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The 3-spined stickleback (Gasterosteus aculeatus) BAC library was constructed by Andrew Stuart in Chris Amemiya’s laboratory at the Benaroya Research Institute at Virginia Mason (BRI) in support of the stickleback genome project. This library was designated VMRC-28. Blood cells were obtained from a single female specimen (#317-2 from Bear Paw Lake in Alaska) that was shown to exhibit the lowest levels of heterozygosity among 16 specimens surveyed at the Broad Institute. Cells were embedded in Incert agarose and high molecular weight DNA was prepared in situ in the agarose. Generation of the library closely followed the cloning approach developed in Pieter de Jong’s laboratory Osoegawa et al., 1998. Partial-digest MboI fragments from the appropriate size fraction were cloned into the pCCBAC-BamHI vector (Epicentre Technologies). The ligation products were then transformed into DH10B (T1 phage resistant) electro-competent cells (Invitrogen). The library has been arrayed into 144 384-well microtiter dishes and has subsequently been gridded onto 22 x 22 cm nylon high-density colony filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones (equivalent to 48 "384-well" dishes), stamped in duplicate. Analysis of 100 randomly selected BACs via pulsed field gel electrophoresis of NotI-digested DNA indicated that the average insert size was around 145 kb. The BAC library construction was funded by NIH grant HG02526 (Chris T. Amemiya, BRI) under the auspices of the NIH Resource Network.
Please note: This library has been used in support of the Stickleback genome project. In the process, many clones have been BAC-end-sequenced. The BAC-end-Sequences (BES) [110,494] can be found in the NCBI Trace Archive using the following query string: center_project="G822"
Segment | 1 | All | Vector | pCC1BAC-BamHI | pCC1BAC-BamHI | Restriction Enzyme | MboI | MboI | DNA Source | blood | blood | Plate Numbers | 1-144 | 1-144 | Plate Count | 144 | 144 | Empty Wells | N/A | N/A | Non-Recombinant Clones | 2764.8 (5%) | 2764.8 (5%) | Non-Insert Clones | N/A | N/A | Recombinant Clones | 52531.2 | 52531.2 | Average Insert Size | 145 Kbp | 145 Kbp | Genomic Coverage | 12X | 12X |
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