Date of Award

8-1-1978

Degree Type

Thesis

Degree Name

M.S.

Department

Department of Biology

First Advisor

Dr. Clarence W. Clark

Abstract

The Cleared Lysate on Sucrose (CLOS) gradient was used in this study as the primary tool in isolating plasmid DNA. The CLOS procedure maintains the "folded configuration" of the bacterial chromosomes at the bottom of the gradients. Studies on the effects of ribonuclease and pronase on the isolation of R6K plasmid deoxyribonucleic acid from Pseudomonas aeruginosa strain RS93 suggested that neither RNase or pronase had any significant effects on the recovery of R6K from RS93. In terms of total percentage recovery of R6K, Triton in the absence of an intermediate ribonuclease (RNase) layer elicited the highest yield of plasmid DNA. Plasmid peak areas from CLOS gradients, presumed to contain R6K, were pooled and subjected to cesium chloride-ethidium bromide (CsCl-EtBr) dye buoyant density gradient analysis. The analysis indicated that only a small percentage of the total molecules recovered banded in the covalently closed circular (CCC) region of the gradient. The majority of the molecules banded in the open circular (OC) and linear regions of the gradient. When plasmid peak areas from CLOS gradients were subjected to alkaline sucrose gradient analysis, there was an absence of recoverable CCC molecules from alkaline gradients. Of the salt concentrations used, 0.5 M NaCl used throughout the CLOS procedure was shown to elicit the highest percentage recovery of R6K from _P. aeruginosa strain RS93.

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