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 Home > Resources > Libraries
  CHORI-222 Drosophila pseudoobscura Sheared/Restriction Digested BAC Library

  
  Background information

The CHORI-222 Drosophila pseudoobscura BAC Library has been constructed in our laboratory by Kazutoyo Osoegawa in collaboration with the Berkeley Drosophila Genome Project directed by Gerald M. Rubin and co-directed by Susan E. Celniker at Lawrence Berkeley National Laboratory. D. pseudoobscura stocks (Tucson 14011-0121.94) were obtained from the Drosophila species stock center, which has relocated to Tuscon, Arizona (http://stockcenter.arl.arizona.edu/). High-molecular-weight DNA was prepared by Roger A. Hoskins at LBNL. The library has been constructed using two methods. For the first segment, D. pseudoobscura agarose embedded DNA was partially digested with a combination of EcoRI and EcoRI Methylase. Size fractionated DNA was cloned into the pTARBAC2.1 vector between the EcoRI sites. For the second segment, the agarose embedded DNA was sheared by repeating freezing and thawing. The fragmented ends were polished by subsequent treatments with Mung bean nuclease and T4 DNA polymerase, respectively. The polished ends were ligated to the blunt-end side of an adapter which has a 3'overhang. Size fractionated DNA was cloned into the pTARBAC6 vector between the BstXI sites. The ligation products were transformed into DH10B electrocompetent cells (BRL Life Technologies). The library has been arrayed into 384-well microtiter dishes and also gridded onto a 22x22cm nylon high-density filter for screening by probe hybridization. The hybridization membrane represents over 18,000 distinct D. pseudoobscura BAC clones, stamped in duplicate.


Provisional data for CHORI-222 Drosophila pseudoobscura BAC Library:

Segments Cloning Vector DNA Restriction
enzyme
Plate Numbers Total Plates Empty Wells Empty Wells (%)
1 pTARBAC2.1 Adult fly EcoRI 1-24 24 28 0.3
2 pTARBAC6 Adult fly sheared 25-48 24 443 4.8

 

Segments
Non-insert Clones (%)
Non-insert clones
Recombinant Clones
Average Insert Size

Redundancy
(Genome size:
120 Mb)
1
8.3
763
45
8,380
156 Kbp
10.9
2
6.3
553
2
8,218
152 Kbp
10.4

 

The total library should represent approximately 21-fold total genomic representation.

Data on the CHORI-222 clone average insert size of Segment 1 and Segment 2 have been determined by Pulsed Field Gel Electrophoresis. Clone size distribution has been plotted graphically. Clone size distribution for each segment has been plotted graphically. While analyzing clones using pulse-field electrophoresis to determine the average insert size, non-insert clones containing a small deleted vector fragment consistent with sucrose resistance were observed. Further in depth characterization of the library is on going in our lab and data will be updated on our web page periodically.

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 filters  are obtainable. For ordering and shipping details, please view the 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).

 

  Reference

Osoegawa K, Vessere GM, Li Shu C, Hoskins RA, Abad JP, de Pablos B, Villasante A, de Jong PJ. BAC clones generated from sheared DNA. Genomics 2007 89:291-9.

 

 

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