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CHORI-1073: Doubled haploid Zebrafish (Danio rerio, Tuebingen line) Fosmid Library |
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The CHORI-1073 Doubled-haploid zebrafish (Danio rerio, Tuebingen line) fosmid library has been constructed by Baoli Zhu and Maxim Koriabine in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital Oakland Research Institute. 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. Genomic DNA was isolated from DNA plugs using GELase (Invitrogen). The purified genomic DNA was sheared by passing through a Hamilton microsyringe, then end-polished by T4 DNA polymerase and T4 DNA polynucleotide kinase treatment. The end-repaired DNA was ligated to pCC1FOS-CHA vector, a derivative of pCC1FOS from Epicentre. This vector was linearized at the unique Eco72I site. The sheared DNA was size purified using a CHEF gel and ligated as recommended by Epicentre’s (vector/insert molar ratio of 10:1). Some of the fosmids (not part of the arrayed library described in the table below) were generated without size purification of the sheared DNA. To reduce the risk of chimeric clones from co-ligation of multiple genomic fragments, a higher molar ratio of vector to insert (30:1)was used in this case. Well over 1 million fosmids have been used for fosmid-end sequencing to aid in the assembly of the zebra fish genome. All ligation products were packaged using Epicentre’s MaxPlax Lambda Packaging Extract. The packaged DNA particles were transfected into the EPI300-T1R strain.
The clones can be found in various public databases using either the "internal" Sanger nomenclature or the "external" NCBI recommended nomenclature. For example, CH1073-15B7 (NCBI nomenclature) corresponds to a clone from the CHORI-1073 Fosmid 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 might be labeled differently. 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 | 2 | 3 | All | Vector | pCC1FOS | pCC1FOS |
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| Restriction Enzyme | N/A | N/A | N/A | N/A | DNA Source | N/A | N/A | N/A | N/A | Plate Numbers | 1-240 | 241-477 | 478-1130 | 1-1130 | Plate Count | 240 | 237 | 653 | 1130 | Empty Wells | N/A | N/A | N/A | N/A | Non-Recombinant Clones | N/A | N/A | N/A | N/A | Non-Insert Clones | N/A | N/A | N/A | N/A | Recombinant Clones | 92160 | 91008 | 250752 | 433920 | Average Insert Size | N/A | N/A | N/A | N/A | Genomic Coverage | N/A | N/A | N/A | N/A |
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