Diphenylamine (DPA) assay:
DNA fragmentation in liver tissue will be carried out according to Perandones et al. (1993).
Principle:
A quantitative method was raised to determining % of fragmentation of known amount of DNA into oligosomal-sized fragments, where DPA form reaction at bond between purines and deoxyribose which is liable forming liberation of inorganic phosphate and provides the substrate (Schiff base) blue color which is measured calorimetrically at 600nm.
Reagents:
1-Hypotonic lysis buffer PH8:
- 0.2% Triton X-100.
- 10 mM Tris.
- 1 mM EDTA.
2-TE (Tris EDTA) buffer:
-10 mM Tris HCl(PH8).
-1 mM EDTA(PH 7.4).
3- 10% Trichloroacetic acid (TCA 10%W/V): 100gm of TCA were dissolved in distilled water and completed up to 1000ml.
4- 5% Trichloroacetic acid (TCA 5%W/V): 50gm of TCA were dissolved in distilled water and completed up to 1000ml.
5- Colorimetric solution:
- 1.5 gm DPA in 100ml glacial acetic acid (98%V/V).
- 1.5 ml sulfuric acid (98%V/V).
- 0.5%V/V of 1.6% acetaldehyde.
Procedure:
1- The liver tissue (40 mg) was mechanically dissociated in 400 µl hypotonic lysis buffer to obtain cell lysate.
2-Incubate at 50 °C for 2.5 hrs.
3-The cell lysate was centrifuged at 13.800xg for 15 minutes.
4-The supernatant (SN), that was contained small fragments of DNA was separated immediately.
5-Acid extraction of DNA:
- The pellet redisolved in TE to be homogenous prior to centrifuge to ensure adequate separation of fragmented DNA from intact DNA.
- Equal volume of 10% TCA was added to both supernatant and pellet.
- Incubation for 15 min at room temperature.
- Centrifugation at 2000rpm for 10 min.
- The periceptate was resuspend in equal volume of 5%TCA.
- Incubation at 100 °C (boiling water bath) for 10 min.
- Centrifugation at 1500 rpm for 15 min.
- The supernatant containing the extracted DNA was left to cool at room temperature.
6-Colorimetric determination of DNA content :
- Two volumes of colorimetric reagent were added to one volume of the extracted DNA (small and large fragments), kept at 30 c overnight till blue color is developed.
7- The developed blue color was quantified spectrophotometrically at 600 nm.
Does anyone have the method in more detail? How can I obtain optical density of supernatant and optical density of pellets?
Check out this link it might answer your question: http://www.cyto.purdue.edu/archive/flowcyt/research/cytotech/apopto/data/chap9.htm
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