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CHORI-75: Double haploid atlantic salmon (Salmo salar) BAC Library |
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The CHORI-75 Double Haploid Atlantic salmon BAC library has been constructed by Boudewijn ten Hallers in Pieter de Jongs laboratory at BACPAC Resources, Childrens Hospital Oakland Research Institute. The preparation of the library followed the cloning approach developed in our laboratory (Osoegawa et al., 1998). Liver tissue was collected from a Double Haploid Atlantic salmon, which was provided by Sissel Jentoft, CIGENE, Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences. Frozen liver tissue was ground under liquid nitrogen into fine powder using pestle and mortar. The powder was suspended in chromatin isolation buffer and further
homogenized using a Dounce homogenizer with a tight pestle (7 ml homogenizer with a tight pestle with 0.0010"-0.0030" clearance). The chromatin suspension was mixed with liquid agarose such that the final agarose concentration was 0.5% and contained a chromatin concentration equivalent with 10 mg of wet tissue per 70 microliters (which is the size of a standard plug in a disposable BioRad plug mold). After solidifying the agarose, the plugs were treated with proteinase K as described previously. The isolated DNA was treated with a combination of EcoRI Restriction enzyme and EcoRI DNA-Methylase to create partially digested genomic DNA with a mean fragment distribution in the 100-300 kb size range. Size fractionated DNA isolated by pulsed-field electrophoresis was cloned using the pTARBAC2.1 vector and transformed into E.coli DH10B (T1 phage resistant). Well-isolated colonies were picked into 384-well microtiter dishes to create the initial BAC array of 624 dishes (also known as "plates"). The clone names follow the NCBI Clone nomenclature rules.
The CHORI-75 BAC library has been created to support the international salmon genome project conducted by an international consortiun consisting of Canadian, Norwegian and Chilean research teams. The library is currently only available to members of this consortium, but will eventually be placed in the public domain. Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI | EcoRI | EcoRI | DNA Source | liver | liver | liver | Plate Numbers | 1-288 | 289-624 | 1-624 | Plate Count | 288 | 336 | 624 | Empty Wells | 3319 (3%) | 2773 (2.15%) | N/A | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | N/A | N/A | N/A | Recombinant Clones | 107273 | 126251 | 239616 | Average Insert Size | 145 Kbp | N/A | N/A | Genomic Coverage | N/A | N/A | N/A |
Acknowledgments:
Please note: This library is not currently available and will only become available after inititial characterization and approval by the consortium.
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