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CHORI-275: Tree shrew (F) (Tupaia belangeri) BAC Library |
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The CHORI-275 “Tree Shrew” BAC library has been constructed by Baoli Zhu, Ph.D., in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital Oakland Research Institute. The preparation of the library followed the general cloning approach developed in our laboratory Osoegawa et al., 1998. Frozen spleen from a female Tree Shrew was provided by the Broad Institute (MIT), 520 Charles Street, Cambridge MA 02141 (Dr. Marcia Lara). The female tree shrew (GSSR # 1005, animal ID# 10448) selected for BAC library construction and subsequent genome sequencing has less heterozygosity compared other available specimens. High-molecular-weight DNA isolated from a spleen cell suspension was embedded in agarose to stabilize the DNA. The embedded DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI methylase then size fractionated by pulsed-field electrophoresis. DNA fragments from the appropriate size fraction were cloned into the pBAC-GMR vector between the two EcoRI sites. The ligation products were transformed into DH10B (T1 resistant) electro-competent cells (Invitrogen). The library has been arrayed into plates 590 384-well microtiter dishes and has subsequently been gridded onto 13 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones, stamped in duplicate. BAC library construction was funded by NIH grant HG025323-03 under the auspices of the NIH-funded BAC Resource Network. Data for CHORI-275 Tree Shrew BAC Library:
Segment | 1 | 2 | All | Vector | pBAC-GMR | pBAC-GMR | pBAC-GMR | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | spleen | spleen | spleen | Plate Numbers | 1-336 | 337-590 | 1-590 | Plate Count | 336 | 254 | 590 | Empty Wells | 2051 (1.59%) | 1287 (1.32%) | 3330 (1.47%) | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | Approx. 3213 (2.49%) | N/A | Approx. 5641 (2.49%) | Recombinant Clones | 123760 | 96249 | 217589 | Average Insert Size | 136 Kbp | N/A | 136 Kbp | Genomic Coverage | N/A | N/A | N/A |
Click here for legend of the previous table.
No clones were found to be non-recombinant after analysis of CHORI-275 using overgo probes specific for the puc19 fragment.
Data on the CHORI-275 clone insert size distribution has been determined by pulsed-field gel Electrophoresis. Clone Size Distribution has been plotted graphically.
The library is available for individual clone orders distributed as agar-stab cultures in a fee structure described on our website "Ordering & Pricing Information" .
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 ([email protected]).
Please direct questions concerning this library to either Pieter J. de Jong.
Ordering & Pricing information
The complete copy of this BAC library is not available. The library is available in several formats. Individual clones, and high-density colony filters for hybridization screening experiments hybridization filter. For ordering and shipping details, please click here.
Reference
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