Observation of DNA by negative staining: phage t7 DNA-capsid complexes

Abstract

A technique has been developed for observing extended nucleic acids in specimens negatively stained with uranyl acetate. This technique has been used to characterize bacteriophage T7 DNA-capsid complexes obtained by: a) disruption of T7 phage using glutaraldehyde treatment; b) isolation from Xysates of bacteriophage T7-infected E. coli. The latter complexes may be in the DNA packaging pathway of T7.A sample of circular, duplex bacteriophage ØX174 DNA (.2 μg/ml) in. 1 M NaCl, .01 M Tris-Cl,. 001 M EDTA, pH 7.4, was mixed with an equal volume of cytochrome c (200 μg/ml) and was incubated for five minutes. This mixture was incubated with a carbon-coated grid for one minute; the grid was washed and was negatively stained with 1% uranyl acetate. An accumulation of negative stain around the DNA-bound cytochrome c reveals the presence of DNA circles (Figure 1). The cytochrome c bound to DNA is observed as an electron transparent region, 40-65 Å in diameter, within the negative stain.