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CHORI-232: Prairie vole (Microtus ochrogaster) BAC Library |
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This BAC library was generated in support of a planned genome project for the Prairie Vole. This species provides preeminent animal model for elucidating the genetic and neurobiological mechanisms governing complex social behavior in vertebrates. See the white paper by Drs. McGraw, Thomas and Young for further details on this model and the justification of the Vole Genome Sequencing Project .
The CHORI-232 BAC Library was generated by Boudewijn ten Hallers and Maxim Koriabine at BACPAC Resources, Children’s Hospital & Research Center Oakland. Procedures followed the cloning approach developed in our laboratory (Osoegawa et al., 1998). Frozen kidney tissue obtained from a male prairie vole (animal ID #102), was kindly provided by Drs. Lisa A. McGraw & James Thomas (Center for Behavioral Neuroscience, Yerkes National Primate Research Center, Emory University, Atlanta). The frozen kidney tissue was ground to a fine powder, which was suspended in chromatin isolation buffer and then embedding in 0.5% InCert agarose. The proteins were removed by a SDS + proteinaseK treatment. The embedded DNA was then partially digested using a combination of EcoRI restriction enzyme and EcoRI methylase. The digested DNA was size fractionated by pulsed-field gel electrophoresis and DNA fragments from the appropriate size fraction were cloned into the pTARBAC2.1 vector between the two EcoRI sites. The ligation products were transformed into DH10B (T1-resistant) electro-competent cells (Invitrogen). The library has been arrayed into 528 “384-well” microtiter dishes and has subsequently been gridded onto 11 nylon high-density filters (22x22cm) for screening colonies by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones (48 dishes), stamped in duplicate.
Acknowledgment: Funding for the construction of the prairie vole library was provided by grants to Larry J. Young from Austism Speaks and the National Institute of Mental Health (1R21MH082225).
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | kidney/brain | kidney/brain | kidney/brain | Plate Numbers | 1-288 | 289-528 | 1-528 | Plate Count | 288 | 240 | 528 | Empty Wells | 2969 (2.68%) | 2558 (2.78%) | 5527 (2.73%) | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | Approx. 1614 (1.5%) | Approx. 1344 (1.5%) | Approx. 2958 (1.5%) | Recombinant Clones | 106009 | 88258 | 194267 | Average Insert Size | 135 Kbp | 142 Kbp | 139 Kbp | Genomic Coverage | N/A | N/A | N/A |
Insert Size Distribution segment 1
Insert Size Distribution segment 2
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