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Nucleic Acids Research Advance Access originally published online on December 6, 2006
Nucleic Acids Research 2007 35(1):e5; doi:10.1093/nar/gkl920
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Nucleic Acids Research, 2007, Vol. 35, No. 1 e5
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

An improved method to identify BAC clones using pooled overgos

Kavitha Madishetty, Pascal Condamine, Jan T. Svensson, Edmundo Rodriguez and Timothy J. Close*

Department of Botany and Plant Sciences, University of California Riverside, CA 92521-0124, USA

*To whom correspondence should be addressed. Tel: +1 951 827 3318; Fax: +1 951 827 4437; E-mail: timothy.close{at}ucr.edu

Received July 6, 2006. Revised September 29, 2006. Accepted October 17, 2006.

Hybridization using overgo probes is an established approach for screening arrayed bacterial artificial chromosome (BAC) libraries. We have improved the use of overgos by increasing the yield of positive clones using reduced levels of radioisotopes and enzyme. The strategy involves labeling with all four radiolabeled nucleotides in a hot pulse followed by a cold nucleotide chase and then extending the exposure time to compensate for reduced specific activity of the probes. The resulting cost savings and reduced human exposure to radiation make the use of highly pooled overgo probes a more attractive approach for screening of BAC libraries from organisms with large genomes.


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