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CHORI-215: Fresh Water Stickleback BAC Library |
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The CHORI-215 Paxton Benthic Stickleback BAC library has been constructed at the Children’s Hospital Oakland Research Institute, BACPAC Resources, by Baoli Zhu in Pieter de Jong’s lab using the cloning techniques developed in our laboratory (Osoegawa et al., 1998). Agarose embedded high molecular weight DNA was prepared from the blood collected from two fishes by Dr. Chris Amemiya in Virginia Mason Research Center at Seattle, WA. To prepare segment 1 of the library, the DNA from a single male (#6) was partially digested with a combination of EcoRI restriction enzyme and EcoRI methylase followed by double size fractionation using CHEF gels. Size fractionated DNA was ligated into the pTARBAC2.1 vector between the EcoRI sites. For segment 2, the DNA from another male (#2) was partially digested with MboI. The size-fractionated DNA was then ligated into the pTARBAC1.3 vector between the BamHI sites. The ligation products were transformed into DH10B (T1 resistant) electrocompetent cells (Invitrogen). The library has been arrayed into 384-well microtiter dishes and gridded onto 4 22x22cm nylon high-density filter for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones, stamped in duplicate. BAC library construction was supported through a sub-contract from a grant awarded to Dr. David Kingsley at Stanford University.
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC1.3 |
| Restriction Enzyme | N/A | N/A | N/A | DNA Source | N/A | N/A | N/A | Plate Numbers | 1-96 | 193-288 | 1-288 | Plate Count | 96 | 96 | 288 | Empty Wells | 324 (0.88%) | 574 (1.56%) | 1176 (1.06%) | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | Approx. 512 (1.4%) | N/A | N/A | Recombinant Clones | 36028 | 36290 | 109416 | Average Insert Size | 148 Kbp | 149 Kbp | N/A | Genomic Coverage | N/A | N/A | N/A |
Content Posted: Sep 30th 2003
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