A technique for the isolation of PlasmidDeoxyribonucleic acid fromPseudomonas Aeruginosa

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ETD Collection for AUC Robert W. Woodruff Library

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http://digitalcommons.auctr.edu/dissertations/390; http://digitalcommons.auctr.edu/cgi/viewcontent.cgi?article=1918&context=dissertations
Yancey, Blonnie Faye
DigitalCommons@Robert W. Woodruff Library, Atlanta University Center
thesis / dissertation description
The Cleared Lysate on Sucrose (CLOS) gradient has been used in these studies as a primary tool in the isolation of plasmid deoxyribonucleic acid (DNA), both sex factor and resistance factor, from Pseudomonas aeruginosa. A concentration of 1.0 M NaCl in the gradient was shown to keep the chromosome in the “folded configuration”. A concentration of 0.05 M NaCl in the gradient was shown to promote chromosomal unfolding.Ten micrograms of ribonuclease (RNase), layered on the gradient, did not affect chromosomal unfolding significantly. Plasmid peak areas from CLOS gradients, presumed to contain the sex factor or resistance factor, were pooled and subjected to Cesium Chloride-Ethidium Bromide gradient analysis. The analysis indicated that only a small percentage of the molecules band in the covalently closed circular (CCC) region of the gradient. The majority of the molecules band in the open circular (OC) and linear regions of the gradient. Contour length measurements of the linear molecules of the FP2 sex factor indicate that their molecular weight is close to the molecular weight of the plasmid, as reported in the literature.