An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA
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Dedon-2009-An Improved Method for Large.pdf
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Author(s)
Barth, Marita C.
Dederich, Debra A.
Dedon, Peter C.
Date Issued
July 2009
Journal
BioTechniques
Publisher
Informa Healthcare
Citation
Barth, Marita, Debra Dederich, and Peter Dedon. “An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA.” BioTechniques 47 (2009): 633-635.
Version
Author's final manuscript
Abstract
A rigorous understanding of the biological function of superhelical tension in cellular DNA requires the development of new tools and model systems for study. To this end, an ethidium bromide–free method has been developed to prepare large quantities of either negatively or positively super-coiled plasmid DNA. The method is based upon the known effects of ionic strength on the direction of binding of DNA to an archaeal histone, rHMfB, with low and high salt concentrations leading to positive and negative DNA supercoiling, respectively. In addition to fully optimized conditions for large-scale (>500 µg) supercoiling reactions, the method is advantageous in that it avoids the use of mutagenic ethidium bromide, is applicable to chemically modified plasmid DNA substrates, and produces both positively and negatively supercoiled DNA using a single set of reagents.
MIT Department
Massachusetts Institute of Technology. Center for Environmental Health Sciences
Massachusetts Institute of Technology. Department of Biological Engineering
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Creative Commons Attribution-Noncommercial-Share Alike 3.0
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DOI of Published Version
https://doi.org/10.2144/000113172
