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VMRC-27: Swordtail fish (Xiphophorus helleri) BAC Library |
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The green swordtail (Xiphophorus helleri) BAC library was constructed by Ayumi Aoki in Chris Amemiya’s laboratory at the Benaroya Research Institute at Virginia Mason (BRI). This library was designated VMRC-27. Blood cells were obtained from a pool of 12 adult “Sarabia” males (DOB 12/1/03, pedigree #8986) obtained from Ronald Walter (Xiphophorus Genetic Stock Center, Texas State University, San Marcos, TX). Blood 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 EcoRI fragments from the appropriate size fraction were cloned into the pCCBACE1 vector (Epicentre Technologies). The ligation products were then transformed into DH10B (T1 phage resistant) electro-competent cells (Invitrogen). The library has been arrayed into 192 384-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 155 kb. The BAC library construction was funded by NIH grant HG02526 (Chris T. Amemiya, BRI) under the auspices of the NIH Resource Network. Segment | 1 | All | Vector | pCC1BAC | pCC1BAC | Restriction Enzyme | EcoRI | EcoRI | DNA Source | N/A | N/A | Plate Numbers | 1-192 | 1-192 | Plate Count | 192 | 192 | Empty Wells | N/A | N/A | Non-Recombinant Clones | 3686 (5%) | 3686 (5%) | Non-Insert Clones | N/A | N/A | Recombinant Clones | 70042 | 70042 | Average Insert Size | 155 Kbp | 155 Kbp | Genomic Coverage | 12X | 12X |
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