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CHORI-245: Formosan muntjac (M) (Muntiacus reevesi) BAC Library |
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The CHORI-245 Formosan muntjac BAC library has been constructed by Maxim Koriabine 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). DNA was isolated from a testicular sample obtained from the Berlin Zoo for a male Formosan Muntjac (Muntiacus reevesi; Berlin Zoo; Born 05/09/2000). The procedure for extracting the chromatin makes use of a published buffer for isolating condensed metaphase chromosomes (for flow cytometry and for cytogenic analysis). This buffer was used to extract DNA from frozen testicular tissue, after grinding the frozen tissue to a fine powder under liquid nitrogen using a pestle & mortar. The agarose-embedded DNA was kindly provided by Dr Harry Scherthan in 2003 (then at the Max Planck Institute in Berlin; currently at the Institute of Radiation Biology in Munich). The high molecular weight DNA was partially-digested with a combination of EcoRI and EcoRI Methylase. Size selected DNA was cloned into the pTARBAC2.1 vector between the EcoRI sites. The ligation products were transformed into DH10B electro-competent cells (T1 phage resistant, Invitrogen). The ligation products were transformed into DH10B (T1 resistant) electro-competent cells (Invitrogen). The library has been arrayed into 528 “384-well” microtiter dishes and has subsequently been gridded onto eleven 22x22cm nylon high-density filters for probe hybridization screening. Each hybridization membrane represents over 18,000 distinct BAC clones (48 dishes), stamped in duplicate. Library characterization was performed by Qing Cao, Teresa Ren and Kazutoyo Osoegawa. BAC library construction was funded by NIH grant HG025323-02 under the auspices of the NIH-funded BAC Resource Network.
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | N/A | N/A | N/A | Plate Numbers | 1-288 | 289-528 | 1-528 | Plate Count | 288 | 240 | 528 | Empty Wells | 3372 (3.05%) | 3330 (3.61%) | 6702 (3.31%) | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | Approx. 1544 (4/277, 1.44%) | Approx. 1572 (4/226, 1.77%) | Approx. 3117 (8/503, 1.59%) | Recombinant Clones | 105676 | 87258 | 192933 | Average Insert Size | 182 Kbp | 156 Kbp | 169 Kbp | Genomic Coverage | N/A | N/A | N/A |
Data on the CHORI-245 clone insert size distribution has been determined by pulsed-field gel Electrophoresis. Clone Size Distribution has been plotted graphically.
No clones were found to be non-recombinant after analysis of CHORI-245 using overgo probes specific for the puc19 fragment.Web page updated: August 27, 2005
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