ahlok2002
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Isolating PC9( lung cancer cell line ) genomic DNA..
I need help here..
i'm looking for the simplest protocol to isolate the PC9 genomic DNA from a culture that already treated with synthetic complex. Then later i
will run the gel electrophoresis to determine the fragmentation of the genomic DNA that may cause by the synthetic complex.
So, my problem is in order to compare the fragmentation of DNA cause by the complex not by the technically mistake, i need a suggestion on the best
isolation protocol.
thanks
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chemoleo
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I am not sure I understand....
Could you elaborate, what is 'synthetic complex'??? What is it supposed to do? Why adding it?
If I understand you correctly, you have two samples of DNA, one is digested with DNases (?), and the other not. Then you compare the two??
Anyway, a few weeks back I already posted a number of professional DNA purification protocols, have a look at http://www.sciencemadness.org/talk/viewthread.php?tid=1496 .
They are scans.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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ahlok2002
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actually i,m working on a anticancer property of an inorganic complex. the synyhetic complex is the inorganic compound (transition metal+amino
acid)that proposed to act like artificial nucleases to cleave the DNA. this cleaving activity of DNA will lead to cell dead at certain concentration.
so.i want to compare the DNA fragmentation betwen the standard nucleases with this synthetic complex to ensure the cleaving pattern. Or manybe you
have any suggestion how i should determine the cleaving activity??
now i try to looking at the suitable protocol that may reduce the error during isolation steps...so i need some advise.
thank you.. chemeleo. i will read up the posts.
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Esplosivo
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Have you decided what restriction enzymes you are going to use? That would be an important step on which the experiment should be built.
The DNA isolation is not that complicated, provided you have access to the apparatus required.
Theory guides, experiment decides.
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chemoleo
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As to the actual DNA purification, use the protocols in the link above. These are the universally used ones. These days, however, people just use
kits, such as Qiagen's miniprep kit.
Anyway - if I understand you correctly - you have two batches of cells, one untreated, and the other treated with your metal complex. On the latter
you suspect double strand cleavage of DNA, which ultimately leads to cell death at high enough concentrations. You want to confirm now that cell death
is indeed induced by double strand DNA breakage, and not by some other toxic mechanism.
Well in this case all I can suggest is this:
Run the two experiments, treated and untreated, at exactly the same conditions.
At time X, start purifying your DNA
Run this on a low density gel (0.5% agarose).
Compare the two, if more or less identical, then no DNA cleavage occurred. If you get smaller fragments, then I guess you have shown that the complex
indeed acts as a nuclease.
However, it may be hard to judge by gel analysis. For instance, the fragments you may get can be random, any size. That means, all you will see (if
you are lucky) is a smear of DNA, where no specific products are produced. With a much smaller amount of very large DNA fragments, compared to the
untreated batch.
In other words, if you DON'T notice any difference between the two gels, this doesn't necessarily mean that your complex has no nuclease
activity!
[Edited on 28-4-2004 by chemoleo]
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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ahlok2002
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True..true..that is what i'm looking for the whole experiment.
i have done the MTT assay for various concentration of the synthetic complex on the PC9, and the result shown that the complex really inhibit the
cell growth and at high enough concentration the complex kill the cell. I have obtain the IC 50 for that complex...now i'm thinking use this
concentration to continue the test that you suggested (isolate the DNA, run the gel).
By looking at the results from the gel, can we suggest how the cell death. Can the ladder of the gel suggest the complex cause cell apoptosis or
necrosis ( let say the pattern of the gel like smear will tell us something )?
From the previous test on the same complex, we found that this complex really cleave the commercial isolated DNA (pBR 322, yeast extract)and the
activity enhanced by adding the H2O2. So we believe that tha cleaving activity will more toward redox activity( involved free species radical) besides
the hydrolytic process. But how i'm going to prove this cleaving activity involves the redox reaction rather that i propose this without any
prove? i got suggestion to add some scavenger like DMSO or ethanol into the standard experiment and compare the with the control. If the rate or the
decelerate of the activity sufficient to suggest that the redox cleaving activity?
thanks chemoleo and Esplosivo...
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In other words, if you DON'T notice any difference between the two gels, this doesn't necessarily mean that your complex has no nuclease
activity!
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chemoleo can tell me more what you want to suggest to me and i really not sure i can get the hint(S)....
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chemoleo
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Hmm, could you please think a little more about your English, it's really hard to understand what you are saying, and reading a post 4 times to
understand what is said is not the way to go 
Anyway...
What is your problem if you have shown the nuclease activity on various plasmids (pbr322) before (there is nothing left to prove!)? I take it this
happens in vitro, and now you want to see whether it happens in vivo, in this immortal lung cancer cell line?
Well there are a number of things you can do.
For one thing, the gel assay would be a start. Compare control vs complex sample, and see whether you get a smear or different fragments.
What I meant is that you won't necessarily see a difference. I have never run a DNA gel on a human unfragmented DNA, so I don't know wht it
looks like. It could be, for instance, that cleavage activity is low (and still deadly), yet not detectable on the gel, when compared to the control.
You can also look at it in a confocal microscope, if you are lucky and cleavage is extensive (the cleavage of the dying/apoptosing cell), you might be
able to see more than 46 chromosomes, but lots of fragments instead. WAy to go!!
But no, the DNA fragment pattern on the gel will NOT tell you whether necrosis or apoptosis will occur. However, apoptosis is more likely, regardless.
Thats normally the mechanism of cell death if the DNA is damaged too much. A microscope will tell you the difference between the two.
Regarding your reactive oxygen species (ROS) - you are introducing a whole new parameter. ROS are, at high concentrations, VERY toxic to cells. They
may induce apoptosis themselves. Be very careful with that. So if you add H2O2, you will definitely get a greater rate of cell death - because you
have TWO toxic compounds acting on the cells rather than just ONE!
In any case, cells naturally produce O2(minus), and the hydroxy radical. Both very toxic, and both contributing to ageing/carcinogenic mutations etc.
So in a highly stressed cell, or a cell that is metabolising rapidly (such as cells dividing), your complex will be more active of course, and thus
will kill those cells. As this is the principle of chemo-therapy (to kill the dividing cells), your complex may be a candidate for chemo-therapy - I hope I will be on the patent 
To show that H2O2 and other oxidising /radical species (define which one?) act on the complex specifically, how about you do a time course experiment,
with your plasmid DNA??
Set up two in vitro experiments - add to both a known amount of plasmid DNA, add a known amount of complex, and add only to one a tiny amount of H2O2.
Incubate for however long it takes, and then run both samples on a gel. You should see an intensity difference between the bands, if H2O2 REALLY DOES
increase cleavage, you will have more fragmentation, which you can see. If you dont see a difference, then increase the dose of H2O2, until you get
really high...and if you still dont see a difference, I think you can exlude the possibility that H2O2 enhances nuclease activity....
[Edited on 30-4-2004 by chemoleo]
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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ahlok2002
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Sorry for my broken English, because I learned English, chemistry and biology 3 years ago…so I still working hard on them. English is one of the
factors cause me hard to express my self.
Back to our discussion….YES, we had obtained the result that the complex shows the nuclease activity on isolated DNA. But what we want to see here
are how the complex act on the DNA in vivol or in vitro and the cleaving mechanisms (hydrolytic or redox).
We are so curious when other cell lines (Hela and HL60) show different sensitivity toward the complex provided same concentration.
Now the problem is how I’m going to explain the different sensitivity of the different cancer cell line???
The reason I can think now may due to H202….because the activity of complex is activated by H2O2, some cancer cell lines may have different level
of H202 in their cytoplasm??(due to fast growth and metabolism rate)
We decided after obtained the mechanisms on how the complex work as nuclease, next we want to proceed to identified whether the complex cause specific
or random cleaving. Later we will determine the cytotocity of the complex on normal cell, hopefully the complex not so toxic on normal cell.
Anyway in experiments we will not get what we always desire….
For now I will think I proceed my works together with adding you suggestions and hope soon I will come back with the discussion..
Really appreciate your effort and patience toward me! Thanks
Learning is not state of being; is a process, is not a destination; is a direction
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