Electron Microscopic Examination of DNA in an Alkaline Sucrose Gradient

The technique of lysing cells on top of an alkaline sucrose gradient prior to sedimentation was first designed to examine single stranded DNA from protoplasts. The lysis of mammalian cells and sedimentation of the released DNA has not been examined thoroughly by EM. The present work describes the ultrastructural characteristics of DNA species found after whole cell lysing and sedimentation of the DNA through an alkaline sucrose gradient.The DNA of H4-II-E rat hepatoma tissue culture cells was prelabelled with H3TdR (luci/ml) for 18 to 24 hours prior to lysis. Cells were layered into a lysing layer (0.1 M EDTA and 0.5 N NaOH, pH 13.25) on top of a 5-202or 5-25% alkaline sucrose gradient (0.01 M EDTA, 0.1 N NaOH and 0.9 M NaCl). After centrifugation the gradients were fractionated from the bottom. The fractions containing the majority of H3TdR were applied directly on grids at pH 8.5 or 12. Samples were stained with uranyl acetate.