DNA fragmentation assay

1. Centrifuge medium at 2500rpm for 10min

2. Add 750 μl of lysis buffer into 60mm cell culture plate

(100mm cell culture plate의 경우에는 1 ml의lysis buffer를 첨가한다.)

3. Stand at 4℃ for 30min

4.Transfer centrifuged cells (pellets : step 1) into 60mm cell culture plate (step2)

5.Scrap with cell scraper

6.Mix cells up and down with pipette (1000P)

7.Transfer to E-tube

8.Centrifuge at 14,000 × g for 20min at 4℃

9.Collect 750 ul of SPNTs to E-tube

10.Add 5ul of 0.5 mg/ml proteinase K

11.Incubate at 50℃ for 2-3hr (possible overnight)

12.Add 750 ul phenol:chloroform:isoamylalcohol (25:24:1)

13.Agitate up and down

14.Centrifuge at 14,000 × g for 10min RT

15.Transfer 500 – 550 ul of SPNTs to E-tube

(tip 끝이 잘라진 것을 사용해야만 한다. 반드시)

16.Add 50 ul of 5 M NaCl and 250 ul of 100% isopropanol

17.Store at -20℃ for 1hr (1시간 이상 보관을 해도됨.)

18.Centrifuge at 14,000 × g for 30min at 4℃

19.Discard SPNTS

20.Wash 500 ul of 70% ethanol

21. Centrifuge at 14,000 × g for 10min

22. Dry on air

23.Add 10 ul of TE-buffer containing 300 ug/ml Rnase A

24.Suspend the pellets

25.electrophorese on 1.5% agarose gel at 50Vfor 1hr 30min

or store at 4℃ (short-term)

20℃ (long-term)