DNA fragmentation analysis protocol - Semi-quantitative method for measuring apoptosis by Richard Pattern (Tufts-New England Medical Center)

Procedure:
  1. Pellet cells
  2. Lyse cells in 0.5 ml detergent buffer: 10 mm Tris (pH 7.4), 5 mm EDTA, 0.2% Triton 
  3. Vortex 
  4. Incubate on ice for 30 min.
  5. Centrifuge at 27000 g for 30 minutes 
  6. Divide supernatants into 2-250 μl aliquots 
  7. Add 50 ul ice cold 5 M NaCl to each followed by vortexing 
  8. Precipitate DNA: Add 0.6 ml 100% EtOH and 150 μl 3 M Na acetate, pH 5.2 place at –80oC freezer for 1 hr. Centrifuge 20,000 g for 20 min and pool DNA extracts by re-dissolving by adding 400 μl total) of extraction buffer (10 mM Tris and 5 mm EDTA). 
  9. Add 2 μl (10 mg/ml) DNase free RNase. Incubate for 5 hr at 37oC. 
  10. Add 25 μl Proteinase K at 20 mg/ml and 40 μl of buffer (100 mm Tris, pH 8.0, 100 mM EDTA, 250 mM NaCl. Incubate overnight at 65oC. 
  11. Extract DNA with phenol, choloform, isoamyl alcohol and precipitate with EtOH.
From Kotamraju et al JBC, 2000 DNA Fragmentation:
A distinctive feature of apoptosis at the biochemical level is DNA fragmentation. This method was used as a semiquantitative method for measuring apoptosis. The culture medium was removed and centrifuged at 3000 × g for 5 min to collect detached cells. Adherent cells were lysed with a hypotonic lysis buffer (10 mM Tris-HCl, pH 8.0) containing EDTA (10 mM) and Triton X-100 (0.5%) and then pooled with pellets made of detached cells. RNA was digested using RNase (0.1 mg/ml at 37 °C for 1 h) followed by proteinase K treatment for 2 h at 50 °C. DNA was extracted with a mixture of phenol, chloroform, and isoamyl alcohol (25:24:1). DNA was precipitated by adding an equal volume of isopropyl alcohol, stored overnight at 20 °C, and centrifuged at 12,000 × g for 15 min at 4 °C. The pellet was air-dried, resuspended in 20 μl tris acetate EDTA buffer supplemented with 2 μl of sample buffer (0.25% bromphenol blue, 30% glyceric acid), and electrophoretically separated on a 2% agarose gel containing 1 μg/ml ethidium bromide and visualized under ultraviolet transillumination.

Dr. Richard Pattern
Tufts-New England Medical Centre
Molecular Cardiology Research Centre
Boston, MA

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