Assay of Nucleosomal DNA Laddering in Apoptotic Cells

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Tony Rook
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Assay of Nucleosomal DNA Laddering in Apoptotic Cells

Please find the following protocol:

Assay of Nucleosomal DNA Laddering in Apoptotic Cells

Overview:

Late in the process of apoptosis, a nucleosomal DNA ladder is generated. This ladder is considered a hallmark of cells undergoing apoptosis.

Procedure:

1. Harvest culture medium containing apoptotic cells by centrifuging medium in a table-top centrifuge at 3,000 rpm for 5 min. Collect 2 to 10 X 106 cells.

2. Carefully aspirate off medium, making sure to leave behind cell pellet.

3. Lyse cells by resuspending the cell pellet in 300 μl Lysis Buffer.

4. Incubate on ice for 10 min.

5. Remix lysate. Remove a 50 μl aliquot and save as a total lysate DNA control sample.

6. Add 1.5 μl of 10 mg/ml RNAse A to give a final concentration of 60 to 70 μg RNAse A per ml.

7. Incubate for 1 hr at 37°C.

8. Add 12.5 μl of 10% SDS so that the final concentration is 0.5%.

9. Add 2 μl of 20 mg/ml Proteinase K so that the final concentration is 150 μg Proteinase K/ml.

10. Incubate for 1 hr at 50°C.

11. Add 1 volume of TE-saturated Phenol.

12. Vortex vigorously.

13. Add 1 volume of SEVAG.

14. Vortex vigorously.

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

16. Transfer the upper, aqueous phase to a new microfuge tube and add 0.1 volumes of 3 M Sodium Acetate.

17. Add 2 volumes of 100% Ethanol.

18. Precipitate the DNA by incubating the sample at -20°C for 1 hr.

19. Pellet the DNA by centrifuging at 13,000 X g for 15 min at 4°C.

20. Decant the ethanol supernatant.

21. Allow the DNA pellet to air dry.

22. Dissolve the DNA in 10 to 20 μl of TE.

23. Add 0.1 volumes Gel Loading Buffer to the DNA.

24. Load sample and a DNA molecular weight marker (see Science Reference Pages) on a 2% agarose gel containing 0.12 μg/ml Ethidium Bromide and made in 0.5X TBE (CAUTION! see Hint #1 and see Protocol on Running DNA on Agarose Gels).

25. Add Ethidium Bromide to 0.12 μg/ml in the 0.5X TBE running buffer as well.

26. Run the gel for about 2 hr at approximately 60 volts.

27. Photograph gel on an UV transilluminator. Expect to see a 200 bp band and a ladder of bands every 200 bp.

Solutions:

TBE (1X)
2 mM EDTA, pH 8.0
89 mM Tris
89 mM Boric Acid

Gel Loading Buffer (10X)
0.1% Xylene Cyanol
0.1 M EDTA, Disodium Salt, pH 8.0
0.1% Bromophenol Blue
50% (v/v) Glycerol

3 M Sodium Acetate, pH 5.0

SEVAG
24:1 Chloroform:Isoamyl Alcohol
(CAUTION Biohazard!)

TE-saturated Phenol
(CAUTION Biohazard!)
Store at 4°C in a dark glass container

20 mg/ml Proteinase K

10% (w/v) SDS

10 mg/ml DNAse-free RNAse A

TE
pH 8.0
10 mM Tris-Cl
1 mm EDTA

Lysis Buffer
0.2% (v/v) Triton-X 100
Prepared in TE

Bioreagents and Chemicals:

Xylene Cyanol
Sodium Acetate
Ethidium Bromide
RNase A, DNase-free
Bromophenol Blue
Tris
Isoamyl Alcohol
SDS
Boric Acid
Triton X-100
Glycerol
Chloroform
Proteinase K
Ethanol
EDTA

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1988