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CHORI-214: Atlantic Salmon BAC Library |
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The CHORI-214 Atlantic salmon BAC library has been constructed by Jim Thorsen and Dr. 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. Sperm was collected from a male Atlantic salmon which was provided from Aqua Gen AS http://www.aquagen.no and embedded in Low-Melting Agarose at a density of 5x107 cells per ml. The agarose embedded high molecular weight DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI Methylase. The DNA fragments were size-fractionated by Pulsed-Field Gel Electrophoresis and DNA fragments from appropriate size fractions were cloned into the pTARBAC2.1 vector between the EcoRI sites. The ligation products were then transformed into DH10B (T1 resistant) electrocompetent cells (Invitrogen). The library has been arrayed into 384-well microtiter dishes and also gridded onto seventeen 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct salmon BAC clones, stamped in duplicate.
Library construction was supported through sub-contracts from grants awarded to Bjorn Hoyheim (MGA-Genetics, Norwegian School of Veterinary Science,) and Professor William S. Davidson (Dean of Science, Simon Fraser University). Carlo Artieri (Simon Frasier University) notified us per email (January 6, 2005) that the standard BAC-end sequencing (BES)primer from the SP6 end (as recommended on our web site)did not work well with the pTARBAC2.1 vector in the salmon library. A new primer designed at Simon Frazier University worked flawlessly to obtain BES data for more than 4,300 BAC clones. The new primer is closer to the vector/insert junction and has the following sequence: actgtggcttgttttacaattt. (Personal Communication).
Provisional data for CHORI-214 Atlantic salmon BAC Library:
Segment | 1 | 2 | 3 | 4 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | MboI | EcoRI/EcoRI Methylase | DNA Source | sperm | sperm | sperm | N/A | sperm | Plate Numbers | 1-288 | 289-576 | 577-816 | 817-960 | 1-960 | Plate Count | 288 | 288 | 240 | 144 | 960 | Empty Wells | 1982 (1.79%) | 2830 (2.56%) | 2975 (3.23%) | N/A | 7787 (2.11%) | Non-Recombinant Clones | 0 (0%) | 5 (0%) | 4 (0%) | N/A | 9 (0%) | Non-Insert Clones | Approx. 1303 (1.2%) | Approx. 3017 (2.8%) | Approx. 2230 (2.5%) | N/A | Approx. 7831 (2.17%) | Recombinant Clones | 107307 | 104740 | 86951 | 55296 | 353013 | Average Insert Size | 189 Kbp | 190 Kbp | 186 Kbp | N/A | 188 Kbp | Genomic Coverage | 6.8X | 6.6X | 5.4X | N/A | 18.8X |
Click here for legend of the previous table.
No clones were found to be non-recombinant after analysis of CHORI-214 using overgo probes specific for the puc19 fragment.
Data on the CHORI-214 clone insert size distribution has been determined by pulsed-field gel Electrophoresis. Clone Size Distribution has been plotted graphically.
Further in depth characterization of the library is ongoing in our lab and data will be updated on our web page periodically. The characterization has also been published (see references below). An expansion of the BAC library using a different partial digest enzyme (MboI) and a different BAC vector is been planned for Fall 2005.
Please direct questions concerning this library to either Pieter J. de Jong or Kazutoyo Osoegawa .
Ordering & Pricing information
The library is available in several formats. Individual clones, and high-density hybridization filter are obtainable. For ordering and shipping details, please view the ordering and pricing information ordering and pricing information page.
Academic and commercial users interested in a copy of the BAC library should contact Pieter J. de Jong (pdejong@chori.org, fax: (510) 450-7924).
References
1)Thorsen J, Zhu B, Frengen E, Osoegawa K, de Jong PJ, Koop BF, Davidson WS, Hoyheim B. A highly redundant BAC library of Atlantic salmon (Salmo salar): an important tool for salmon projects.BMC Genomics. 2005,6:50.
2)Ng SH, Artieri CG, Bosdet IE, Chiu R, Danzmann RG, Davidson WS, Ferguson MM, Fjell CD, Hoyheim B, Jones SJ, de Jong PJ, Koop BF, Krzywinski MI, Lubieniecki K, Marra MA, Mitchell LA, Mathewson C, Osoegawa K, Parisotto SE, Phillips RB, Rise ML, von Schalburg KR, Schein JE, Shin H, Siddiqui A, Thorsen J, Wye N, Yang G, Zhu B. A physical map of the genome of Atlantic salmon, Salmo salar.Genomics. 2005 Jul 15
Updated: August 22, 2005
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