How can I degrade dna extract? I used H2O2 with concentrations ranging from 50 mM to 500 mM but the band on gel only appeared thinner than control but not smeared, i used U.V lamp and still had the same result.Do I have to induce damage on cultured cells rather than pure DNA extract? In my research i normally used COMET or cytokinesis block, but they would be time consuming and expensive andf a bit advanced for the undergards I have.
...I'm hypothesizing but
I would definitely try the H2O2 on the cells. (also UV)
It is oxidizing the DNA (so forming adducts not directly breaks), which in an intact cell system leads to ss and ds breaks but on isolated DNA where no enzymes are, the damage is not recognized so no DNA break induced.
It will depend on the cells and you will need to set the concentration
but with 100-500µM you might need to wait several hours before harvesting the cells..... with lower concentrations you could treat over night (afternoon and harvest next day).
If there is an opportunity to induce DNA fragmantation within the cultured cells, the cheapest way is to induce apoptosis using calcium. You can incubate your cells in complete growth media supplemented with additional CaCl2 over night which would cause an apoptosis, during which DNA is usually cut. According to my experience, CaCl2 solution should be used at final concentration 2 mM, but it can differ between the cell lines, so maybe you will have to test several concentrations to reach the result.
Another importatnt thing for sample preparation is to collect the growth medium even if you use adherent cells, because during apoptisis it can detach.
According to the literature, DNA fragmentation can be induced with the help of calciun even in cell extracts, but the procedure is more complicated, see http://www.jbc.org/content/279/34/35564.full
Maybe the UV radiation source you have will also work on cells, because it also causes apoptisis during which DNA is cut by enzymes. It should be far more efficient than radiation itself.
Good luck!
Dear Yassmine,
the cheapest and quick method is using crude DNA extracts from yeast or from onion and observe the floated fluffy nucleoproteins which are visible by naked eye to students and you can transfer small amounts of these, mix with ethidium bromide,apply it to wells of 1% agarose gel . Run for 50 minutes and you also can recognize smeared bands of DNA under UV. You can do the last step prior to lab and keep the gel refrigerated for demonstration next day or even on deep freeze for more than 2 weeks.
Thank you all for your helpful answers, I guess it is a necessity to induce damage in a culture..which needs expensive reagents and culture hood and incubator...I tried induction NA damage on whole blood using CaCl2, H2O2 and U.v light..however for some reason, the blood clumped when I added ethanol during extraction (I use spin columns)
aDear Dr najat..yes i already practice gel electrophoresis with students..however my question was if there was a way to induce breaks in DNA after its extraction, so they can visualize the smear
Ultrasound sonication is good choice for fragmentation of extracted DNA.
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