Apoptosis DNA Fragmentation Analysis Protocol
Licia Miller Product Manager
The DNA of apoptotic cells is cut into fragments of about 200 base pairs under the action of caspase-activated nuclease (CAD), forming DNA ladders. By detecting DNA fragmentation during apoptosis, the degree of apoptosis can be evaluated.
This experiment uses agarose gel electrophoresis to detect DNA fragmentation. If more rapid or quantitative analysis is required, the use of a TUNEL kit for flow cytometry or microscopy analysis can be considered.
Stage 1 Cell collection and lysis
1. Collect cell samples, wash once with PBS, and remove the culture medium.
2. Add 0.5 mL of lysis buffer (10 mM Tris, pH 7.4; 5 mM EDTA; 0.2% Triton X-100) to each cell sample.
3. Vortex and incubate the sample on ice for 30 minutes to fully lyse the cells.
4. Incubate the lysed samples at 27,000 × g for 30 min under 4℃. Collect the supernatant and divide it into two aliquots of 250 µL each.
5. Add 50 µL of ice-cold 5 M NaCl to each supernatant and vortex gently to mix.
Stage 2 DNA extraction and purification
1. Add 600 µL of ethanol and 150 µL of 3 M sodium acetate (pH 5.2) to each sample and mix gently. Keep the samples at -80 °C for 1 hour.
2. Centrifuge at 20,000 × g for 20 min and carefully discard the supernatant.
3. Combine the DNA extracts and resuspend the pellet in 400 µL extraction buffer (10 mM Tris, 5 mM EDTA).
4. Add 2 µL of 10 mg/mL DNase-free RNase to the combined DNA extracts and incubate at 37 °C for 5 hours to fully remove RNA.
5. Add 25 µL of 20 mg/mL proteinase K and 40 µL of buffer (100 mM Tris, pH 8.0; 100 mM EDTA; 250 mM NaCl) and incubate overnight at 65°C to remove proteins.
6. Extract the DNA with phenol/chloroform/isoamyl alcohol (25:24:1) and remove the upper aqueous phase. Then precipitate the DNA with ethanol and carefully discard the supernatant.
Note: The DNA precipitate is relatively loose at this time, so be sure not to disturb the precipitate when removing the supernatant.
7. Air-dry the DNA pellet at room temperature.
Stage 3 DNA agarose gel electrophoresis
1. Resuspend the DNA pellet in 20 µL TAE buffer and add 2 µL sample buffer (containing 0.25% bromophenol blue and 30% glycerol).
2. Prepare 2% agarose gel (containing 1 µg/mL ethidium bromide).
3. Separate the DNA by electrophoresis.
4. Use a UV transilluminator to observe the DNA ladder structure and take photos to record the results. In the agarose gel electrophoresis diagram, the appearance of a DNA ladder structure of about 200 bp indicates that the cells have undergone apoptosis.
Negative control: Cell samples without induced apoptosis should have no obvious DNA ladder structure.
Positive control: Cells can be treated with an agent known to induce apoptosis (e.g., H2O2) as a positive control.
Precautions
● Operating environment: The entire experimental process must be carried out at low temperature to prevent DNA degradation.
● Reagent selection: Ensure that all reagents and consumables are RNase-free and DNase-free.
● Sample handling: Samples should be handled gently and avoid violent shaking to avoid affecting DNA integrity.
● Electrophoresis conditions: Adjust the electrophoresis time and voltage according to the size of the DNA fragments, usually 100 V for 30-60 min.
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