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Posts posted by Bluenoise

  1. I'm sorry I wish I had answers to your questions. I feel that whatever conclusion you will find will come have to come from within.

    However if I may I'd like to make a suggestion. This is in the understanding that you don't like satisfying this dopamine fix, but knowing that you can't help it. I'd like to suggest you find alternative methods to satisfy this craving. Try Sports, Playing music, Dancing, Writing. Goto the gym and go crazy for 1 and a half hours. I say this because your currently methods will only drag you deeper into your problems. You need something real, something clean. Not something that will further distance you from reality or damage your mind.

  2. I think the biggest problem with this argument is that most discoveries rely on discoveries in seemingly unrelated projects and fields. It's not just a matter of doing a lot of research on something to discover it. That's a very naive perception. To discover some thing the supporting environment and infrastructure has to be there. Notice how many major discoveries are often happen to be reported by the different groups at the same time? It's because the world was finally in a state where science had progressed to the point where the discovery had become virtually inevitable. In essence science had become primed for the discovery.

    Now because research is a search for something unknown and there are often many hypothesis or none at all how can you accurately say "we have the tools and background knowledge necessary to archive this goal". Well you can't! Because you can't know exactly what the necessary precursors are till you're there.

    Really often you must have some minimal understanding of the system as whole to progress an individual aspect of it.


    Take cancer research for example. There has been progress definatley, life spans are slowly increasing. However to the volume of money that has been pumped into it is disproportionately large. I'd argue that the lack of solid progress on finding a "cure" isn't due to such an thing being completely impossible (at least in every sense of the word). But more so due to the fact that underling technology and infrastructure doesn't exist to allow for such a discovery. However unfortunately most government and populations are very poorly educated about what it takes to allow a scientific discovery to happen. So we have a disproportionate large sum of funding going directly to cancer research as apposed to research aimed at biologically engineering techniques that are desperately needed to advance the ultimate goals. Even physics and chemistry are very important to this field. Take biophotonics without this cancer research would go nowhere. Or what about all those underfunded organic chem labs designing synthesis techniques that are ultimately used to build the small molecules that we currently used to treat cancer?


    Even take your example of quantum computing. It's likely a matter of not having proper material and techniques that is due to the slow rate of implementation, not a matter of a lack of brain power

    It's really sad how little credit the guys working on the techniques and design and engineering get compared to the ones that apply it.


    I say you put a disproportionate amount of funding into one thing. And you go nowhere fast.

  3. Actually It's starting to look more and more unlikely that the beneficial effect derived from ingestion of anti-oxidants is due to their ability to absorb free-radicals. Or at least entirely due to this.

    The premises that this is why they are good for you is purely derived from conjecture.

    For example:


    There is excellent evidence that eating fruits and vegetables is good for you.

    Fruits and vegetables contain large amounts of anti-oxidants.

    Thus eating large amounts of anti-oxidants must be good for you.

    And anti-oxidants protect your body from harmful oxidation.


    I'm pretty sure I'm not the only one who can see how seriously this logic is flawed.


    Actually most studies done where people have ingested a large dose of a single anti-oxidant (usually a vitamin) have show either no health benefit or deleterious health effects. Vitamin E is a good example.


    If I were to put my money on a beneficial effect for anti-oxidants I'd say that the health benefits derived from them is more so due to chemical genetic interaction.

    For example:

    Antioxidant A is structurally similar to Toxin B. This causes the body to respond by alteration of gene express to protect the body from Toxin B. However as Toxin B isn't present the observable effect from Antioxidant A is a health benefit. This is because it basically steps up the bodies defenses.


    More evidence is gaining to support this second hypothesis. For example there is a powerful antioxidant Resveratrol found in wine which is believed to be partially responsible for the health benefit derived from red wine (though other compounds are now being shown to be equally if not more important). Studies have shown that daily doses of this anti-oxidant promote longevity in Yeast, mice, and drosophila. Also resveratrol has been show to be a regulator of mitochondrial proliferation.

    Other work has linked the maintenance of mitochondria to longevity. Actually the expression of mitochondrial proteins and their integrity is very strongly linked to many health benefits. Also the inverse is true as well, where mitochondrial defects are show to be very deleterious.

    And guess what's the natural function of Resveratrol? It's a Toxin. Well it's not toxic to us, it's an anti fungal present in the skins of fruits.


    I think part of the problem occurs from the fact that we sloppily group all these compounds antioxidants; Which causes people to assume this antioxidant property is what's beneficial about these compounds. However there is really no evidence that they protect in such a manner in-vivo.


    While the evidence is still very scarce about their actual mode of action. I feel pretty confidant in saying that I believe it is far more complex than that of a sponge for free radicals.

    I cringe every time someone gives this explanation. As at most it can only be partial correct.


    (Anti-oxidant proteins are a strong exception to what I've said however. I'm referring to small molecules here)

  4. Unfortunately I probably cannot afford that one.


    Yeah... ...also unless you do a massive amount of sequencing is far more affordable to just outsource it. (Not to say that you don't do a large amount of sequencing of course)


    We have two fairly new sequencers in our lab just sitting around gathering dust. It's just not worth the time, effort, and resources to do it ourselves when we can send it down the hall for $8 a run. Though it would be quicker to do it myself.

  5. It's amusing how this post came back to the top again after so long. Well anyways I might as well give an update.

    The DNase I method worked like a charm. I've been able to create a very functional library with amazingly high sequence diversity and genetic coverage. I estimate that 1uL of my 120 uL library has about 10,000 fold genetic coverage of the bacterial genome that I'm working with.


    Thanks all for your suggestions.

  6. microbial engineering? I didn't even know there were departments specifically for this. You may fair better if you search for biomedical engineering programs first. Then pick a supervisor that is catering towards the microbial engineering side of it.

    At almost every school microbial engineering is likely to be a sub-discipline in biomedical engineering programs. This would likely explain your difficulty in finding a microbe-engineering department.

  7. Oh no I would definatley not recomend radiolabling for PCR.

    But in this instance where there might be a band that can't be visualized with the current method it does offer far better sensitivity than anything else. Plus basically nothing can interfere with its detection.

    Personly I only ever radiolable when working with very small amounts or under conditions where I can't visualize any other way. Radiation is a pain in the ass.


    I was just thinking, the OMP does not happen to bind to biotin, does it?


    Now wouldn't that be ironic.

  8. CharonY has a good point. How heavy is this protein? Could it not just be getting stuck in the well?


    Also what mechanism are you using for chemiluminesent detection?

    There's a good possibility that binding of the protein to the DNA molecule is blocking the chemiluminescent signal. If you've constructed the potential binding site to close to the biotin linker you might have some issues with steric hinderance. If this is the case likley adding a longer spacer arm for the biotin linkage or additional nucleotides, maybe necessary.


    You should stain with Etbr to see if there is maybe a ghost band that you can't see. Or if your lab is equiped radiolable the oligo, this can be done even with a 5' biotin as you can add additional nucleotides before the biotin modification.

    I'm a bit confused as to why you would go through all the trouble of this chemiluminescent visualization.

  9. Ah, well too late to answer then. But if memory serves in denatured gels the speed is roughly equal (would make sense if both are linear ss, of course). However in PAGE (as opposed to agarose) 22bp can be resolved.

    You can clean-up DNAseI treated RNA with columns w/o phenol extraction, btw.


    Well in the end it didn't matter. The labled UTP i used was a bit old and didn't give a great signal.

    Gave it another try. I DNase treated it. Purified on a QIAGEN nucl. removal kit., dephosphorylated, rephosphorylated.

    Got a nice gel. a little degraded sample but good resolution. Was able to purify RNA from it.


    The gel took 12 hours to run... lol

  10. Hey. So I was a bit of an idiot and forgot to digest away my template after transcription. Yeah figures. So as I've already added loading dye to my transcrption I can't exactley add DNase I to it now, so I got to run it on a gel first.

    I guess my quesiton is: is there a significant enough difference in the migration rates of DNA and RNA on denaturing PAGE (urea) that I will be able to differentiate two peices of about equal length. Lets say the RNA is 350 bp and the DNA 22 bp longer than that. If I do get two bands differentiating between the two shouldn't be difficult since only my RNA is radio-labled.


    Some one please say yes.


    I really don't want to have to DNase I treat it after purifying my RNA from the gel... cuz phenonl:chloroform extraction is a pain in the ass.

  11. He's basically making a comparison of the structural of viral capsids to various geometries. Like dodecahedrons etc... Along with this is the use of two different mathimatical methods to generate these geometries. Either the use of simple mathametics or a quantum harmonic ocillator.

    A fairly interesting read actually. Lot of great images.

    Though the math is definatley a little past what I've become accustomed to.

  12. couldn't you put the gene into a plasmid, UV mutate it, and then plonk the plasmid into the bacteria?


    that way, only the gene of interest would be mutated.


    Sure but you'd still have a working copy on the chromosome.


    I'm having trouble seeing what exactley is the goal here. If you just want to knock out a particular gene well I'd go about it as CharonY suggested. Unless it's an essential gene, things get a little more complicated then.


    If you want to look at the effect of specific mutations, then knock it out and complimet it with mutatated versions on plasmids.

  13. I'm a bit confused at to what causes the clumping in the tail. Shouldn't you get an even smeer or gradient or at the most bands. This is a one dimensional seperation is it not?


    What does this sort of assay involve. To me it looks like you have some hight molecular weight DNA "stuck" in a high density gel and you digest/degrade the DNA and perform electrophoresis simultaniously. Is this an assay for rate of degradation of the DNA?

  14. Touchdown PCR is very simular to what I'm getting at except unfortunatley it still deals with rather short sequences. I think I'll just have to see how it works out before coming to any conclusions. I figure letting the temp drop from 95-65 in 20-30 mins should be adequate. I hope these elevated temperatures for long periods dont degrade my DNA too badly.

    Hmmm anyone know the temperature that DNA starts to anneal at? I know 92-95 is a good bet for denaturing it all but there is likely a gap below that where it doesn't start to anneal.


    Actually now that I think of it the best approach maybe to use chemical conditions. I can denature my DNA in 0.25 N NaOH and then slowly renature it with the addition of drop wise 3M NaOAc to a 1/10 final concentration. Though the problem here is that I don't exactley know how quickly it anneals under such conditions.

  15. Let say you fragment genomic DNA into around 500bp fragments. Now lets say you denature these fragments by heat or whatever. How would you go about ensuring that as many fragments as possible anneal to their complementary strand. Now obviously as you'll likely have 1000s of times genomic coverage each fragment will have 1000s available that it can consider complementary but it will have about 10 000 - 100 000 times that are not considered complementary.


    Conventional wisdom suggest that the best solution is to just mix them together in a buffer of moderate salt concentration heat a beaker to around 95C drop the tube containing the oligos in and let it slowly cool to at least 65C.


    However is there a better way? I reallised that annealing oligos is a pretty simple thing. However, usually one considers small oligos and rarely more than 2-4 species. Finding the optimum match for each becomes more tricky when you have long ones and a 100 000 different species.

    Maybe the best thing to do it just greatly increase the time it take the temperature to drop?

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