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[#x02013]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 microg) 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.
Barth
Marita
M
Department of Biological Engineering and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
Dederich
Debra
D
Dedon
Peter
P
D000818:Animals; D002021:Buffers; D002645:Chickens; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000097:blood; D004278:DNA, Superhelical / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D019800:Phenol / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D002021:Buffers; D004278:DNA, Superhelical; D005971:Glutamates; D006657:Histones; D011994:Recombinant Proteins; D019800:Phenol; D004247:DNA; D004264:DNA Topoisomerases, Type I