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CHORI-258: Owl monkey (M) (Aotus hybrid) BAC Library |
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The CHORI-258 Owl Monkey BAC library has been constructed by Baoli Zhu in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital Oakland Research Institute. The preparation of the library followed the cloning approach developed in our laboratory (Osoegawa et al., 1998). DNA was isolated from kidney cells obtained from a male owl monkey obtained from The Squirrel Monkey Breeding and Research Resource (SMBRR, Department of Comparative Medicine, University of South Alabama, Mobile, AL36688). The local SMBRR identification number for this animal is 85286. Upon subsequent karyotype analysis, it was discovered that the animal was a likely F1 hybrid with a maternal chromosomes from Aotus nancyma (Ma cytotype I) and paternal chromosomes possibly from from A. azarai (Ma cytotype VI) (or A. infulatus). Standard and G-band karyotype analyses indicate a 2n=51,XY chromosomal constitution, not compatible with the original species assignment of Aotus nancyma (2n=54). Gender determination by PCR, and sequencing of a partial control region of mitochondrial DNA (P. Gagneux) support the cytogenetic results in that this male monkey appears to carry a nancymaae-like mitochondrial DNA, but an azarai-like fusion of the Y chromosome to an autosomal chromosome. The karyotype analysis was kindly performed at the Cytogenetics Laboratory, Center for Reproduction of Endangered Species, Zoological Society of San Diego. A skin biopsy and a blood sample have been sent to the Coriell Cell Repositories to establish cell lines as part of the Integrated Primate Biomaterials and Information Resource (IPBIR) initiative. High-molecular-weight DNA was isolated from a kidney cell suspension prepared in a Dounce Homogenizer and was embedded in agarose to stabilize the DNA. The embedded DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI methylase and size fractionated by pulsed-field electrophoresis. 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 will be arrayed into 528 384-well microtiter dishes and will subsequently be gridded onto 11 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones, stamped in duplicate. BAC library construction was funded as part of the NIH-funded BAC Resource Network by a grant from NIH (#HG025323-02).Provisional data for CHORI-258 Owl monkey (M) (Aotus hybrid) BAC Library:
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI | EcoRI | EcoRI | DNA Source | adult male kidney | adult male kidney | adult male kidney | Plate Numbers | 1-288 | 289-528 | 1-528 | Plate Count | 288 | 240 | 528 | Empty Wells | 2808 (2.54%) | 1794 (1.95%) | 4602 (2.27%) | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | N/A | N/A | N/A | Recombinant Clones | 107784 | 90366 | 198150 | Average Insert Size | N/A | N/A | N/A | Genomic Coverage | 5X | 5X | 10X |
Click here for a legend of the previous tables.
The CHORI-258 clone average insert size has been determined from a sample of 172 colonies prior to the arraying of the BAC library. Pulsed Field Gel Electrophoresis of NotI digested BACs with appropriate size standard was used for size determination. The library will be available as individual clones, and high-density hybridization filters once the arraying, library duplication and initial characterization is complete.
Individual clones identified after screening can be obtained as stab cultures at a cost of $80 per clone in addition to a $35 set-up charge per shipment. Shipping will be done by Federal Express and charged to the recipients account number.
With respect to the scheduling of shipments of arrayed library copies, we would like to make you aware of our policy for array distribution.
Please visit the ordering information page if you want to place an order.
Academic and commercial users interested in a copy of the BAC library should contact Pieter J. de Jong ([email protected], fax: (510) 450-7924). For hybridization membranes, please contact BACPAC Resources ([email protected]).
Date initially posted: June 9, 2003. Revision dates: June 9, 2003; June 19th, 2003; November 24, 2004
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