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CHORI-264: White-throated Sparrow (Zonotrichia albicollis) BAC Library |
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The CHORI-264, White-throated Sparrow, BAC Library was generated by Boudewijn ten Hallers and Maxim Koriabine in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital & Research Center Oakland. Procedures followed the cloning approach developed in our laboratory (Osoegawa et al., 1998). Frozen kidney tissue obtained from a female sparrow, sample ID:822, was kindly provided by James W. Thomas, PhD in the Department of Human Genetics at Emory University School of Medicine, 615 Michael St., Suite 301, Atlanta, GA 30322. The frozen kidney tissue was ground to a fine powder, resuspended in chromatin isolation buffer then embedding in 0.5% InCert agarose. The proteins were removed by a detergent/proteinaseK treatment and the resulting high molecular weight DNA partially digested using a combination of EcoRI restriction enzyme and EcoRI methylase enzyme. The DNA was size fractionated by pulsed-field gel electrophoresis and DNA fragments from the appropriate size fraction were cloned into the pTARBAC2.1 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 528 (384-well) microtiter dishes and has subsequently been gridded onto 11 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones, stamped in duplicate.
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | kidney/brain | kidney/brain | kidney/brain | Plate Numbers | 1-288 | 289-528 | 1-528 | Plate Count | 288 | 240 | 528 | Empty Wells | 3106 (2.81%) | 2464 (2.67%) | 5570 (2.75%) | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | Approx. 430 (1/252, 0.4%) | Approx. 395 (1/227, 0.44%) | Approx. 828 (2/479, 0.42%) | Recombinant Clones | 107056 | 89301 | 196354 | Average Insert Size | 146 Kbp | 141 Kbp | 144 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-264 using overgo probes specific for the puc19 fragment.
Data on the CHORI-264 clone insert size distribution has been determined by pulsed-field gel Electrophoresis. Clone Size Distribution has been plotted graphically.
Questions related to this library should be sent to Pieter J. de Jong (pdejong@chori.org, fax: (510) 450-7924).
Posted: September 19, 2008
Revised: October 22, 2008
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