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Nucleic Acids Research Advance Access published online on December 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm974
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© 2007 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

Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining

Michael B. Smeaton1, Paul S. Miller1, Gary Ketner2 and Les A. Hanakahi1,*

1Department of Biochemistry and Molecular Biology and 2Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD, 21205, USA

*To whom correspondence should be addressed: Tel: +1 443 287 2515/2326; Fax: +1 410 955 2926; Email: lhanakah{at}jhsph.edu

Received August 28, 2007. Accepted October 18, 2007.

The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1–5 x 109 cells. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host–virus interactions and analysis of mutant cell lines.


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