Isolation and purification of DNA from mass cultures of the free-living amoeba

Abstract

A method is described for the isolation of a purified, highly polymerized DNA from the free-living amoeba. Proteinase K, RNase T1, RNase (Aspergillus clavatus), phenol extraction, and density gradient centrifugations are important steps in obtaining satisfactory DNA recoveries. The DNA preparations contained no protein and were slightly contaminated with RNA (orcinol-positive material). Since about 70% of the orcinol-positive material was not affected by KOH treatment and since only a small amount of it remained after DNase treatment, it is suggested that the most of the orcinol-positive material is not RNA but is some structural carbohydrate. The high degree of polymerization of the isolated DNA was estimated by comparing its buoyant density in CsCl with those found previously for nuclear DNA. © 1984 Carlsberg Laboratory.