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CHORI-216: Nigerian (M) Frog (Xenopus tropicalis) BAC Library |
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The CHORI-216 Nigerian frog Xenopus tropicalis BAC library (segment 1) has been constructed by Dr. Michael Nefedov 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). Agarose embedded DNA was obtained from Chris Amemiya at the Virginia Mason Research Center as part of a collaborative effort to create the Xenopus tropicalis BAC library. The DNA was isolated at VMRI from a Nigerian male frog in the 7th generation of inbreeding (N7). The frog was part of the (Stock 248 F7A2) population maintained by Robert M. Grainger, University of Virginia. Gender was determined by visual analysis. DNA was isolated by embedding erythrocytes in agarose. Agarose embedded DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI Methylase and the fragments were size-fractionated by pulsed field electrophoresis. DNA fragments from the appropriate size fraction were cloned into the pTARBAC2.1 vector between the EcoRI sites. The ligation products were then transformed into DH10B (T1 resistant) electro-competent cells (Invitrogen). The library has been arrayed into 384-well microtiter dishes and also gridded onto four 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct Xenopus BAC clones, stamped in duplicate.
Library characterization will be performed by Qing Cao and Dr. Kazutoyo Osoegawa.
An extension of the library using a different restriction enzyme (MboI) for generating genomic fragments has recently been generated by Yuko Yoshinaga (April 2004) and is being expanded further to reach a total genomic redundancy of 15X for the entire CHORI-216 BAC library.
BAC library construction was funded by NIH grants HG01165-07SI and HG025323-01 (Pieter J. de Jong, CHORI) and HG02526-01 (Chris T. Amemiya, VMRC).
Provisonal data for CHORI-216 Nigerian frog Xenopus tropicalis BAC library:
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC1.3 |
| Restriction Enzyme | EcoRI/EcoRI Methylase | MboI | N/A | DNA Source | blood | blood | blood | Plate Numbers | 1-171 | 172-432 | 1-432 | Plate Count | 171 | 261 | 432 | Empty Wells | 1952 (2.97%) | 1752 (1.75%) | 3704 (2.23%) | Non-Recombinant Clones | 0 (0%) | N/A | 0 (0%) | Non-Insert Clones | Approx. 1147 (1.8%) | Approx. 20679 (21%) | N/A | Recombinant Clones | 62565 | 77793 | 162184 | Average Insert Size | 162 Kbp | 119 Kbp | N/A | Genomic Coverage | 5.8X | 5.3X | 11X |
Click here for legend of the previous table.
No clones were found to be non-recombinant after analysis of CHORI-216 using overgo probes specific for the puc19 fragment.
Data on the CHORI-216 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 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.
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 & pricing information page.
Academic and commercial users interested in a copy of the BAC library should contact Pieter J. de Jong ([email protected], fax: (510) 450-7924).
Reference:
Unavailable!
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