Internucleosomal DNA fragments can readily be detected by agarose gel electrophoresis. The small DNA fragments that are isolated from apoptotic cells are well resolved by gels. The simple and reproducible detection of internucleosomal DNA fragmentation has made it a biochemical hallmark of apoptosis. However, it should be noted that some cell lines (e.g. MCF-7, DU145) show almost no DNA ladders, although they show the typical morphological changes of apoptosis (see Section 1).
Protocol 2. DNA extraction and standard agarose gel electrophoresis
Equipment and reagents
• horizontal agarose gel electrophoresis chamber
• electric power supply
• ethidium bromide solution
• UV transilluminator
• TAE (40 mM Tris-acetate, 1 mM EDTA) or TBE buffer (45 mM Tris-borate, 1 mM EDTA)
• DNA sample loading solution (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficol in water
1. After harvesting the cells, samples are washed with phosphate-buffered saline (PBS) and pelleted by centrifugation.
2. The cell pellets are lysed in a solution containing 50 mM Tris-HCI (pH 8.0), 20 mM EDTA, 10 mM NaCI, 1% (w/v) sodium dodecylsulfate (SDS).
3. Lysates are incubated sequentially with 20 jjig/ml RNase A at 37°C for 60 min and 100 fjig/ml proteinase K, at 37°C for 3-5 h.
4. Lysates can then be applied directly on 1.2% agarose gel in TAE or TBE buffer in a horizontal gel support. In order to get clear DNA ladder images, it is important to optimize the cell number and the volume of cell lysis buffer because too many cells make the sample viscosity too high, and reduce the gel resolution.
5. Pour 1.2% agarose gel in TAE or TBE buffer in a horizontal gel support. Insert the comb and let the gel solidify.
6. Load samples to the gel wells, and perform electrophoresis.
7. Stain gels with 5 n,g/ml ethidium bromide in TAE, TBE buffer, or water for 30 min. Resolved DNA fragments can be viewed on a UV transilluminator box and photographed.
8. Alternatively, DNA can be extracted from cell lysates by standard phenol/chloroform/isoamyl alcohol extraction procedures if sharp images cannot be obtained with whole-cell lysates.
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