细胞凋亡提取DNA ladder的操作规程
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Apoptosis-DNA ladder Assay
1. Collect culture media, add 1 ml of trypsin to cell mono1ayer on 100-mmol/L dishes, scrape the cells, harvest cells (culture media and cell monolayer) by centrifugation (2,500 rpm, 5 min), and wash cell pellets with undefined PBS
2. Add 100 μl of lysis buffer (1% NP-40 in 20 mmol/L EDTA, 50 mmol/L Tris-HCl, pH 7.5) for 10 sec. [“Lysis of cells”]
3. Centrifugation (3,000 rpm, 5 min) and obtain supernatant
4. Add 10 μlof 10% SDS solution to pooled supernatant (final : 1% SDS), treat with 10 μl of 50 mg/ml RNase A (final 5 μg/μl) and incubate for 2 h at 56 ℃.
5. Add 10 μl of 25 mg/ml Proteinase K (final 2.5 μg/μl) and incubate for 2 h at 37 ℃
6. Add 1/2 vol. (65 μl) of 10 M ammol/Lonium acetate
7. Add 2.5 vol. (500 μl) of ice-cold ethanol and mix thoroughly
8. Stand for 1 h in – 80 C freezer (“ethanol precipitation”)
9. Centrifuge for 20 min at 12,000 rpm, wash the white pellet with 200 μl 80% ice-cold ethanol and air-dry for 10 min at room temperature
10. Dissolve the pellet with 50 μl of TE buffer
11. Determination of DNA concentration (Abs 260) and 2% agarose gel electrophoresis of the same concentration of DNA (about4 μg)
