Perhaps you want to share a few more details about what you are planning to do? Then people can answer more specifically. I know and have used a number of different protocols, depending on the application. Roughly said, you can use fragmentation via sonication or even pipetting or enzymatic treatments.
No actually, I am treating cancer cell lines with drug which is causing lethality in cell lines. Hence, I want to validate my result by looking for DNA fragmentation which is feature of apoptosis.
There are usually 2 protocols for DNA fragmentation studies. The first one is designed to get only fragmented DNA, the second one aims to purify all DNA from cells.
The protocol for fragmented DNA includes precipitation of nuclei with intact DNA; fragmented DNA comes to supernatant.
We used to use protocol as follows:
1. Collect cells. Both floating and adherent ones.
2. Lyse cells in 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.2% Triton X-100. 500 mkl per 1-2 Petry dishes, 20 min on ice.
3. Spin down at appr. 5000 g (you can try more gs). So, here you separate nuclei from fragmented DNA.
4. Take supernatant, add into it proteinase K up to 0.1 mg/ml. Incubate at 50 dgs C at least 4 h.
5. Add 1 volume (appr. 500 mkl) of PCI (phenol:chloroform:isoamyl alcohol 25:24:1).
6. End-over-end at least 10 min, RT.
7. Spin down, take the aquatic (upper) phase that contains DNA+RNA.
8. Add appr. 1/10 of volume 3М Na Acetate, рН 5.2, add >0.7 of volume of isopropyl alcohol (or 2.5 volumes of ethanol).
9. Leave samples at -20 dgs C for 1-2 h. Or overnight. Or forever. In our hands samples were OK for more than 3 years.
10. Centrifuge 10-15 min at 12000-15000 rpm in a microcentrifuge.
11. Discard supernatant, add 0.5-1 ml of 70% ethanol.
12. Centrifuge 5-10 min at 12000-15000 rpm in a microcentrifuge.
13. Discard supernatant, air dry pellets. Sniff samples. If the don't smell anymore (that usually takes 5-10 min), dissolve pellets in 10-15 mkl of TE (http://en.wikipedia.org/wiki/TE_buffer).
14. Add 1 mkl of RNase A (10 mg/ml stock solution). Incubate 30-60 min at 37 dgs C.
15. Add 6x loading buffer, go to electrophoresis.
There are many protocols to purify total DNA from cells. They differ in first steps (lysis and getting rid of proteins), but subsequent steps are very similar to above steps 5-14.
Please try Comet Assay if you have a long term on going project.
Your exact response is depend to what you're planning to test. For instance in case of apoptosis assay you can use DNA laddering instead of TUNEL test which both assay the fragmented DNA. You need just ordinary electrophoresis equipment to get this test done. Results will be similar to ladder if your DNA was fragmented.
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