An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA
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 p...
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| Format: | Article |
| Language: | en_US |
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Informa Healthcare
2011
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| Online Access: | http://hdl.handle.net/1721.1/67309 https://orcid.org/0000-0003-0011-3067 |
| _version_ | 1811090053635506176 |
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| author | Barth, Marita C. Dederich, Debra A. Dedon, Peter C. |
| author2 | Massachusetts Institute of Technology. Center for Environmental Health Sciences |
| author_facet | Massachusetts Institute of Technology. Center for Environmental Health Sciences Barth, Marita C. Dederich, Debra A. Dedon, Peter C. |
| author_sort | Barth, Marita C. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T14:31:36Z |
| format | Article |
| id | mit-1721.1/67309 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:31:36Z |
| publishDate | 2011 |
| publisher | Informa Healthcare |
| record_format | dspace |
| spelling | mit-1721.1/673092022-10-01T21:37:03Z An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA Barth, Marita C. Dederich, Debra A. Dedon, Peter C. Massachusetts Institute of Technology. Center for Environmental Health Sciences Massachusetts Institute of Technology. Department of Biological Engineering Dedon, Peter C. Dedon, Peter C. 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. National Cancer Institute (U.S.) (NCI; grant no. CA072936) National Cancer Institute (U.S.) (NCI; grant no. CA110261) National Cancer Institute (U.S.) (NCI; grant no. CA103146) National Institute of Environmental Health Sciences (ES002109) National Defense Science and Engineering Graduate Fellowship 2011-11-29T20:38:31Z 2011-11-29T20:38:31Z 2009-07 2008-12 Article http://purl.org/eprint/type/JournalArticle 0736-6205 http://hdl.handle.net/1721.1/67309 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. https://orcid.org/0000-0003-0011-3067 en_US http://dx.doi.org/10.2144/000113172 BioTechniques Creative Commons Attribution-Noncommercial-Share Alike 3.0 http://creativecommons.org/licenses/by/3.0/ application/pdf Informa Healthcare PubMed Central |
| spellingShingle | Barth, Marita C. Dederich, Debra A. Dedon, Peter C. An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA |
| title | An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA |
| title_full | An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA |
| title_fullStr | An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA |
| title_full_unstemmed | An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA |
| title_short | An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA |
| title_sort | improved method for large scale preparation of negatively and positively supercoiled plasmid dna |
| url | http://hdl.handle.net/1721.1/67309 https://orcid.org/0000-0003-0011-3067 |
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