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A 5x genome coverage bovine BAC library: Production, characterization, and distribution

Zhu, B and Smith, JA and Tracey, SM and Konfortov, BA and Welzel, K and Schalkwyk, LC and Lehrach, H and Kollers, S and Masabanda, J and Buitkamp, J and Fries, R and Williams, JL and Ross Miller, J (1999) 'A 5x genome coverage bovine BAC library: Production, characterization, and distribution.' Mammalian Genome, 10 (7). 706 - 709. ISSN 0938-8990

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A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin ( PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

Item Type: Article
Subjects: Q Science > QH Natural history > QH426 Genetics
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Jim Jamieson
Date Deposited: 23 Oct 2014 11:27
Last Modified: 17 Sep 2019 11:15

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