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onlyus
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[*] posted on 16-1-2005 at 20:06
gel electrophoresis


I was just wondering if anyone had a good idea about a chemical/substance that I could run through my gel box besides DNA(since it happens to be kinda pricey). It also needs to be able to be seen as it runs through the gel. thanks!

[Edited on 18-1-2005 by onlyus]
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Hermes_Trismegistus
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[*] posted on 17-1-2005 at 09:38
pigments...(from flowers)






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[*] posted on 17-1-2005 at 09:51


You can extract DNA from severlas sources its very easly done with household chemicals I remember there is a thread on that somewhere.
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[*] posted on 17-1-2005 at 10:14


Essentially you could purify many types of of small to medium sized compounds, the density of the gel (or rather the percentage of agarose) would have to be adjusted for the molecular size of the compound.

The other problem of course is detection -how do you know where your product is, or when it comes out at the other end of the gel? With DNA it's easy, as there is a dye that binds to DNA reversibly (ethidium bromide). This way the bands on the gel can be visualised

Further, the compound would have to be ionic, in order to be moved by the electric field.




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neutrino
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[*] posted on 17-1-2005 at 10:35


Wouldn’t DNA have to be chopped up with restriction enzymes before electrophoresis?
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[*] posted on 17-1-2005 at 11:48


Yes, and THOSE are expensive to say the least.

Perhaps amino acids from proteins can be used, but I dont know how to split proteins up, and what dye would be used?

I know scientists do "amino acid sequencing" and thats gotta be sometning like this....




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chemoleo
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[*] posted on 17-1-2005 at 12:23


I don't know how you get to your statements....but NO, DNA does NOT have to be cleaved with restriction enzymes to be run on a gel & purified. Why would you want to do this? RE's are used for cloning and such, and for genetic mapping etc. I
DNA can be run on a gel at nearly any size. If it is genomic DNA, such as derived from onion cells (See the DNA purification thread in the biochem. Section) it is fairly large in terms of the no. of base pairs (i.e. 300000), so a low density (i.e. 0.5%) gel is run. If the pieces are short (i.e. a 300 base pair PCR product, uncleaved with RE's), then a 1.5-2% is run.
RE's are just a tool to create sticky ends. Or it is indeed used to chop up large pieces of DNA, search i.e. RFLP analysis if you wish.

Anyway, amino acids are probably too small to be separated by gel electrophoresis, although special gel agents are sometimes used . Reverse phase columns (HPLC) with special resins or gas chromatography are normally employed.




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[*] posted on 17-1-2005 at 12:39


onlyus, I cannot understand your post quite well. Do you need a 'marker' or 'tracker dye' so to know when to stop running the gel or are you thinking about some other substitute for DNA for your electrophoresis apparatus? If your problem is the latter, just use a charged dye, such a methyl orange. Owing to the presence of a sulfonic acid group the latter would move towards the positive terminal and therefore 'creating' a band, which would mimmick the DNA band. Amino acids could be run and stained with ninhydrin but since they are very small they would probably cluster up and produce one longish band. (Hydrolysing proteins with dilute mineral acid would give the respectice amino acids - a note for Jome) Usually though amino acids are separated using isoelectric focusing and not electrophoresis, although the principle behind both is quite similar.

If the problem is the former you will require more expensive and complicated chemicals. First of you will need what are known as Molecular Weight Markers which are basically segments of DNA of different masses which are known, and with which one can 'estimate' the size of an unknown by comparing to these standards. If what you need is a dye which moves during electrophoresis and gives you an indication of when to stop then you need 'Tracker Dyes', such as Bromocresol green, bromophenol blue, xylene cyanol, amongst others.




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chemoleo
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[*] posted on 17-1-2005 at 12:46


Ah, I think I understand him now. Presumably he wants to test his gel box, using agarose, but doesnt want to use DNA cus it's expensive and needs carcinogenic ethidium bromide.
In this case, just use bromophenol blue (edit - which Esplosivo mentions), an inexpensive dye that is normally included with a DNA sample, to mark the solvent front - i.e. it runs along with very small pieces of DNA. THis can be seen nicely on the gel too.




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[*] posted on 17-1-2005 at 12:47


I’m guessing that he’d want clear, distinctive bands, which is why I assumed the enzymes would be needed.
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[*] posted on 17-1-2005 at 12:50


Could be out my depth here.
Can you make your own gel or can you add something like fluorocene or some fluorescent agent to it, similar to thin layer chromatography where fluorocene is added and a lot of stuff quenches the fluorocene and show up as dark areas under 254 (I think they are sold as germicidal lamps or UV curing lamps, I am not sure of that without checking, not the purple glow in the dark lights, they only pick up fluorescent stuff)

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[*] posted on 17-1-2005 at 12:59


mick, that is the same thing as using ethidium bromide although I don't known if it would work. Ethidium bromide is a fluorescent dye which intercalates between bases of nucleic acids (DNA, RNA). On being exposed to UV this EtBr gives off light indicating the position of the nucleic acid on the agarose. It's main disadvantage, apart from being expensive, is that it is highly carcinogenic as previously mentioned by chemoleo. Of course there are other, maybe lesser used stains, which can show up the presence of nucleic acid on the agarose although such 'blotting' procedure requires a lot of time and more chemicals which are not easy to come by.



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[*] posted on 17-1-2005 at 13:23


I was thinking if you have got a bit of kit but you cannot run it up to speed run it in reverse. If you cannot find out what is glowing find out what is not.
mick
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[*] posted on 17-1-2005 at 17:01


Mm, using my great mind powers, I sense that this "onlyus" actually wants to use a gel box to seperate and observe at least 2 different chemicals. ;)

In other words, they do a synthesis of some hypothetical chemical, and then use the gel box to either analyze the the products, or seperate the products. That would be an interesting use for a gel box. But not knowing much about gel boxes myself, I'm have no good ideas.

Just an idea. (Ok, ok, so I referred onlyus to this site :P)

So any reasonably simple reactions that would produce products seperable in this way? (I suppose that there wouldn't have to be a reaction, the 2 chemicals could be mixed together)

Cyrus




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onlyus
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[*] posted on 17-1-2005 at 20:22


Cyrus, you read my mind! an assortment of chemicals/dies/pigments/whatever can be run through agarose that is visible would be nice. i need a substitute for DNA, not just a marker. And no, I do not want anything carcinogenic, thanks anyway:D
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[*] posted on 18-1-2005 at 06:52


If you are in to experimenting I would try using a mixture of bromophenol blue and methyl orange in glycerol. Make the agarose dense, i.e. increase the amount of agarose per volume of water. This might work out although I don't know if they might move together due to similar 'speed' of movement. If this does not work I remeber that on a certain site they mentioned that a mixture of pigments used as food colouring could be used to seperate the different constituents although I am not sure if this works either. Hope this helps.



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