Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
CONTRIBUTOR:
OVERVIEW:
PROCEDURE:
The Laboratory of Qin Chen at the University of Arizona
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.
1. Remove the medium from a cell culture flask (see Hint #1).
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 (see Hint #2) by resuspending them in 300 μl of TE/Triton Buffer.
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 (see Hint #3).
8. Centrifuge the remaining lysate at 13,000 X g for 15 min at 4°C (see Hint #4).
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 (see Hint #5) and incubate the sample for 1 hr at 37°C.
11. Add 12.5 μl of 10% SDS (see Hint #6).
12. Add 2 μl of 20 μg/ml Proteinase K (see Hint #7).
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 (see Protocol ID#1265).
22. Photograph the gel under UV illumination (CAUTION! See Hint #8 and Hint #9)
SOLUTION:
5 M NaCl
20 μg/ml Proteinase K
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
REAGENTS AND CHEMICALS:
Tris
EDTA
Sodium Chloride
Proteinase K
Triton X-100
Boric Acid
Xylene Cyanol
Ethidium Bromide
Agarose
Isopropanol
Bromophenol Blue
SDS
Sucrose
RNase A
PROTOCOL HINTS:
1. The supernatant medium will contain apoptotic cells.
2. Begin with approximately 2 to 10 X 106 cells.
3. This sample can be used for a total lysate DNA sample.
4. This pellets the cell debris and whole nuclei.
5. The final RNase concentration should be 60 to 70 μg/ml.
6. The final SDS concentration should be 0.5%.
7. The final Proteinase K concentration should be 150 μg/ml.
8. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
9. Expect to see a 200 bp band and a ladder of fragments that increase in 200 bp increments.