Abstract
Dark-field electron microscopy allows high resolution imaging of super- coiled DNA. Plasmid molecules isolated from Halobacterium halobium were spread by basic monolayer techniques with cytochrome c. They were subsequently mounted onto thin indirectly deposited carbon films and positively stained with ethanolic uranyl salts. A Siemens Elmiskop 102 was operated at 10 000 and 40 000 times magnification using tilted and hollow cone dark-field illumination respectively. Micrographs obtained (Fig. 1) allowed contour length determination by tracing molecules and direct counting superhelical turns. This led to calculation of the superhelical density of large plasmids. The contour length of supercoiled and of relaxed circular molecules which were contained within the same spreading solution and thus photographed from the same grid were evaluated by projection of micrographs and tracing with a digitizer connected to a Wang 2200 calculator. The values obtained are (49.1 ± 2.5) μn and (52,7 ± 2.2) μm for superhelical and relaxed circles respectively.
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