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CHORI-73: Doubled-haploid Zebrafish (Tuebingen line) (Danio rerio) BAC Library |
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The CHORI-73 Doubled-haploid zebrafish (Danio rerio, Tuebingen line) 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). The source DNA was derived from a single double-haploid male fish provided by Dr. John H. Postlethwait from the Institute of Neuroscience, University of Oregon. DNA was isolated after agarose-embedding of chromatin obtained from the frozen fish. The procedure for extracting the chromatin makes use of a published buffer for isolating condensed metaphase chromosomes (for flow cytometry and for cytogenic analysis). This buffer was used to extract DNA from the single fish, after the frozen fish had been grounded up to a fine powder using a pestle & mortar, under liquid nitrogen. The high molecular weight DNA was partially-digested with a combination of EcoRI and EcoRI Methylase. Size selected DNA was cloned into the pTARBAC2.1 vector between the EcoRI sites. The ligation products were transformed into DH10B electro-competent cells (T1 phage resistant, Invitrogen). The library has been arrayed into 390 384-well microtiter dishes and also gridded onto 8 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct zebrafish BAC clones, stamped in duplicate. The clones can be found in various public databases using either the "internal" Sanger nomenclature or the "external" NCBI recommended nomenclature. For example, CH73-15B7 (NCBI nomenclature) corresponds to a clone from the CHORI-73 BAC library found in microtiter dish (384-well type) #15, at the intersection of row "15 and column "7". The same clone in the "internal" Sanger nomenclature is labeled as: "zH15B7". When ordering clones from BACPAC Resources Center (BPRC, @CHORI) using the online ordering system, please use the NCBI nomenclature.
Overviews of the BAC and fosmid libraries used for the zebrafish sequencing and mapping projects can be found at the Sanger "Fingerprinting web page" and the Sanger "Zebrafish Libraries" page.
The name of zebrafish strain used for the BACs is "Tue" (Tuebingen). The characteristics of this fish line are described in the following paper: Rauch, G.-J., Granato, M. and Haffter, P. (1997). A polymorphic zebrafish line for genetic mapping using SSLPs on high-percentage agarose gels. Technical Tips Online T01208.
Segment | 1 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | whole animal | whole animal | Plate Numbers | 1-390 | 1-390 | Plate Count | 390 | 390 | Empty Wells | 1992 (1.33%) | 1992 (1.33%) | Non-Recombinant Clones | N/A | N/A | Non-Insert Clones | Approx. 389 (1/380, 0.26%) | Approx. 384 (1/380, 0.26%) | Recombinant Clones | 147379 | 147384 | Average Insert Size | 110 Kbp | 110 Kbp | Genomic Coverage | 9.5X | 9.5X |
Click here for a legend of the previous tables.
Data on the CHORI-73 clone insert size distribution has been determined by pulsed-field gel Electrophoresis. Clone Size Distribution has been plotted graphically.
No clones were found to be non-recombinant after analysis of CHORI-73 using overgo probes specific for the puc19 fragment.
Further (in depth) characterization of the library is on going in our laboratory and data will be updated on our web page periodically.
The library is available as library plates, individual clones, and high-density hybridization sets (see Filter Availability)
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 .
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