Apoptosis Assay using DNA Laddering

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Tony Rook
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Apoptosis Assay using DNA Laddering

Please find the following protocol:

Apoptosis Assay using DNA Laddering

Overview:

This protocol is designed to determine if nucleosomal DNA laddering, a phenomenon sometimes associated with apoptosis, occurs in cultured cells. Activation of endogenous endonucleases resulting in double strand breaks in nuclear DNA at 180 to 200 bp-multiple intervals is a common event in the apoptotic cascade.

Procedure:

1. Remove the medium from a cell culture flask. The supernatant medium will contain apoptotic cells.

2. Centrifuge the medium at 1,000 X g for 5 min.

3. Aspirate the supernatant from the tilted tube while watching for the cell pellet.

4. Lyse the cells by resuspending them in 300 μl of TE/Triton Buffer. Begin with approximately 2 to 10 X 106 cells.

5. Incubate the cell suspension on ice for 10 min.

6. Gently swirl the tube to resuspend the cells again.

7. Set aside 50 μl of this cell lysate. This sample can be used for a total lysate DNA sample.

8. Centrifuge the remaining lysate at 13,000 X g for 15 min at 4°C. This pellets the cell debris and whole nuclei.

9. Transfer the low molecular weight DNA-containing supernatant to a fresh microcentrifuge tube.

10. Add 1.5 μl of 10 mg/ml RNase A and incubate the sample for 1 hr at 37°C. The final RNase concentration should be 60 to 70 μg/ml.

11. Add 12.5 μl of 10% SDS. The final SDS concentration should be 0.5%.

12. Add 2 μl of 20 μg/ml Proteinase K. The final Proteinase K concentration should be 150 μg/ml.

13. Incubate the sample for 1 hr at 50°C.

14. Add 0.1 volume (26.5 μl) of 5 M NaCl.

15. Add 1 volume (256 μl) of -20°C Isopropanol.

16. Incubate the sample on ice for 10 min.

17. Centrifuge at 13,000 X g for 15 min at 4°C.

18. Decant the supernatant by inverting the tube on a tissue.

19. Dissolve the DNA pellet in 10 to 20 μl of TE Buffer.

20. Add sufficient Gel Loading Buffer to make the final concentration 1X.

21. Run the sample on a 2% Agarose Gel with a 1 Kb DNA ladder as a marker.

22. Photograph the gel under UV illumination. Expect to see a 200 bp band and a ladder of fragments that increase in 200 bp increments.

Solutions:

10% (w/v) SDS

10 mg/ml RNase A.................................................DNase-free

TE/Triton Buffer...................................0.2% (v/v) Triton X-100
......................................................10 mM Tris
.............................................................pH 8.0
......................................................1 mM EDTA

TE Buffer...............................................................10 mM Tris
...............................................................1 mM EDTA

Gel Loading Buffer (10X)...........0.05% (w/v) Bromophenol Blue
.................0.05% (w/v) Xylene Cyanol
...............................................1X TBE
.............................60% (w/v) Sucrose

TBE Buffer.................................................2 mM EDTA, pH 8.0
.............................................................89 mM Tris
....................................................89 mM Boric Acid

5 M NaCl

20 μg/ml Proteinase K

Bioreagents and Chemicals:

Triton X-100
Xylene Cyanol
Boric Acid
Ethidium Bromide
Agarose
Isopropanol
Bromophenol Blue
SDS
RNase A
Sucrose
Tris
EDTA
Proteinase K
Sodium Chloride

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1989