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VMRC-29: tsetse fly (Glossina m.morsitans) BAC Library |
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The Tsetse fly (Glossina morsitans morsitans) BAC library was constructed by Garnet Navarro and Andrew Stuart in Chris Amemiya’s laboratory at the Benaroya Research Institute at Virginia Mason. This library was designated VMRC-29. Tsetse fly specimens were obtained from Dr. Serap Aksoy, Yale University School of Public Health. These specimens were reared on antibiotic-containing media in order to reduce gut microflora. Nuclei were prepared from whole pupae (~50) using a dounce homogenizer and several rounds of centrifugation. The nuclei were then 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 resistant) electro-competent cells (Invitrogen). The library has been arrayed into 144 384-well microtiter dishes and has subsequently been gridded onto 22x22cm 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 125 randomly selected BACs via pulsed field gel electrophoresis of NotI-digested DNA indicated that the median insert size was around 152 kb and the average insert size was 138 kb (the descrepancy is due to a small fraction of smaller insert-containing clones in the library, i.e., clones < 100 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: The following data is provisional pending further characterization.
Segment | 1 | All | Vector | pCC1BAC | pCC1BAC | Restriction Enzyme | EcoRI | EcoRI | DNA Source | N/A | N/A | Plate Numbers | 1-144 | 1-144 | Plate Count | 144 | 144 | Empty Wells | N/A | 0 (0%) | Non-Recombinant Clones | 3317 (6%) | 3317 (6%) | Non-Insert Clones | N/A | N/A | Recombinant Clones | 51979 | 51979 | Average Insert Size | 138 Kbp | 138 Kbp | Genomic Coverage | 13X | 13X |
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