mbo J “1446712 15: 15961606. Schanda P, ” Kupce E, Brutscher B SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33: 199211. 10 October 2011 | Volume 6 | Issue 10 | e25981 Chromatin Immunoprecipitation: Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Relebactam Sheared DNA, and Analysis via PCR Pamela D. Schoppee Bortz1, Brian R. Wamhoff1,2 1 Cardiovascular Division, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia, United States of America, 2 Robert M. Berne Cardiovascular Research Center, University of Virginia Health System, Charlottesville, Virginia, United States of America Abstract The “quantitative”ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the MockChIP supernatant via the phenol-chloroform-isoamyl alcohol method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR. Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest. Citation: Schoppee Bortz PD, Wamhoff BR Chromatin Immunoprecipitation: Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR. PLoS ONE 6: e26015. doi:10.1371/journal.pone.0026015 Editor: Yamini Dalal, National Cancer Institute, United States of America Received July 25, 2011; Accepted September 15, 2011; Published October 25, 2011 Copyright: 2011 Schoppee Bortz, Wamhoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided by the National Institutes of Health and by an American Heart Association Scientist Development Grant to BRW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected]; [email protected] Introduction Molecular biologists commonly use the chromatin immunoprecipitation assay is a tool to study protein-DNA interactions in healthy and diseased biological systems. As a result, numerous variations of the original approach to ChIP are present within the peer-reviewed literature and on molecular biology protoco