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 Home > Resources > Libraries
  RPCI - 11 Human Male BAC Library

  

The RPCI-11 Human Male BAC Library (Osoegawa et al., 2001) was constructed using improved cloning techniques (Osoegawa et al., 1998) developed by Kazutoyo Osoegawa.  The library was generated by Kazutoyo Osoegawa.  Construction was funded by a grant from the National Human Genome Research Institute (NHGRI, NIH) (#1R01RG01165-03).  This library was generated according to the new NHGRI/DOE "Guidance on Human Subjects in Large-Scale DNA Sequencing".

Male blood was obtained via a double-blind selection protocol.  Male blood DNA was isolated from one randomly chosen donor (out of 10 male donors) and partially digested with a combination of EcoRI and EcoRI Methylase.  Size selected DNA was cloned into the pBACe3.6 vector between the EcoRI sites. For Segment 5, the same male donor DNA was partially digested with MboI, size selected, and ligated into the pTARBAC1 cloning vector at the BamHI 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 22x22cm nylon high density filters for screening by probe hybridization.

The RPCI Human Male BAC Library:

Segment

Cloning Vector

DNA

Plate Numbers

Total Plates

Total Clones

Empty Wells (Total)

1

pBACe3.6 (1) 

Male 1-288 288 108,499 2,093

2

pBACe3.6 (1) Male 289-576 288 109,496 1,096

3

pBACe3.6 (1) Male 577-864 288 109,657 935

4

 pBACe3.6 (1)  Male 865-1152 288 109,382 1,210

5

pTARBAC1 (2) Male 1153-1440 288 106,763 3,289

Total Library

    1-1440 1440 543,797 9,163

1: donor DNA EcoRI partially digested
2: donor DNA MboI partially digested

Segment Empty Wells (%) Non-Recombinant Clones (Total) Non-Recombinant Clones (%) Insert Size (average) Genomic Coverage
1 1.9 approx. 1800 1.7 164 Kbp 5.8X
2 1.0 approx. 550 0.5 168 Kbp 6.0X
3 0.8 approx. 1100 1.0 181Kbp 6.7X
4 1.1 approx. 1100 1.0 183Kbp 6.8X
5 3.5 approx. 530 0.5 196Kbp 6.9X
Total Library 1.7 approx. 5080 0.9 178 Kbp 32.2X

click here for a legend of the previous tables.

The average insert size has been determined by Pulsed Field Gel Electrophoresis analysis of clones randomly chosen from plates from each segment.

Reference:

1. Osoegawa, K., Mammoser, A. G., Wu, C., Frengen, E., Zeng, C., Catanese, J. J., de Jong, P. J. (2001) A Bacterial Artificial Chromosome Library for Sequencing the Complete Human Genome, Genome Research, Vol. 11, Issue 3, 483-496, March 2001 (Reprints available upon request)

2. Osoegawa, K., Woon, P.Y., Zhao, B., Frengen, E., Tateno, M., Catanese, J.J, and de Jong, P.J. (1998) An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries, Genomics 52, 1-8; Article # GE985423. (Reprints available upon request)

The library is available for individual clone orders, distributed as agar-stab cultures at a not-for profit cost. See our webpage "Ordering & Pricing Information" for the costs .

The BAC library has been gridded onto 22x22cm positively charged nylon filters for hybridization screening purposes. Each filter contains 36,864 colonies which represents 18,432 independent clones spotted in duplicate in a 4x4 clone array. Clones can be identified through screening by purchasing "high-density colony" hybridization filters or by utilizing our "fee for service" "Library Screening Services" . For questions about clones and hybridization membranes, please contact BACPAC Resources (BACPACorders@chori.org).

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 (pdejong@chori.org, fax: (510) 450-7924). For hybridization membranes, please contact BACPAC Resources (BACPACorders@chori.org).

 

 

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