Chemical Forums
Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: gera19 on February 21, 2006, 05:48:37 AM
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The extraction of DNA
What is the purpose of the salt water? I think that the neutralized DNA molecules tend to aggregate one another but not sure..
Also, what is the purpose of the liquid soap? Does it got to do with its amphipathic structure and it possessing a structure very similar to a phospholipid molecule?
When the soluble DNA comes into contact with the alcohol where the 2 layers of liquid meet,the DNA gets dehydrated and precipitates into the alcohol layer.What molecules/components are left in e bottom layer of liquid?
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Salted water gives you the oppurtunity to perform a density gradient centrifugation, where you separete nucleic acids from proteins, because DNAs have higher density.
We use detergents for solubilising cell membranes.
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for the 3rd question, is the answer liquid soap molecules(greasy parts) and proteins?
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Well, by that time, I think you should have already deproteinised the preparation. So, I think the answer is just liquid soap molecules(greasy parts).
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Salted water gives you the oppurtunity to perform a density gradient centrifugation, where you separete nucleic acids from proteins, because DNAs have higher density. We use detergents for solubilising cell membranes.
This sounds dodgy. As far as I know, the salt solution (sodium acetate usually, plus a protease) is meant to precipitating the proteins inside the solution.
density gradient centrifugaration is used for segregating the different organelles.
soap solution actually "open up" the cell by forming intermolecular bonds with the plasma membrane. The hydrophobic head of the phospholipid bilayer bonds more favourable to the soap molecules than to bond among themselves. coincidentally, the cell nucleus (which contains the DNA) has a phospholipid double layer acting as a membrane (aka nuclear envelope). The soap solution "opens" the nuclear envelope, facilitating the DNA to enter the solution.
DNA is water-soluble, but is relatively insoluble in alcohol. By virtue of that intermolecular bonding between DNA is stronger that of intermolecular bonding between DNA and alcohol, the DNA molecules tend to form intermolecular bonds with each other, thus aggregating together inside ethanol. The aggregation of the DNA is observed as precipitation of DNA in the alcohol solvent.
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density gradient centrifugaration is used for segregating the different organelles.
Trust me: plasmid DNA, in particular, is subject to density gradient ultracentrifugation through ceasium chloride.
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Trust me: plasmid DNA, in particular, is subject density gradient ultracentrifugation through ceasium chloride.
This sounds dodgy. As far as I know, the salt solution (sodium acetate usually, plus a protease) is meant to precipitating the proteins inside the solution.
Lily: what salt is being used in the context of your question?
Apparently, 2 different salt solutions are being used in DNA extraction.
DNA extraction consist of the following steps:
1. "open up" the cells using soap water.
2. use aq. CsCl2 for centrifugration to segregate different components.
3. extract the crude DNA from the mixture
4. add aq. sodium acetate and protease to remove any protein in the crude DNA by precipitation. (purification)
5. filter away the precipitate, and add excess alcohol to the filtrate.
6. the DNA precipitates in excess alcohol, which can be removed by filtration.
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Ok, here is the procedure I've followed so far.
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Generally density gradient ultracentrifugation is not used in purifying DNA. Yes, ultracentrifugation is a useful technique for studying DNA, but it is completely unrelated to DNA purification.
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i'm very confused...besides my frd gave me these answers to e bio qs :1st qns) the salt water one.. salt
> water causes protein n carbohydrate in the cell to be precipitated...so they
> can be removed from the cell...den can isolate DNA..
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> 2nd qns) soap. soap will destroy the plasma membrane of the cell. it breaks
> down the lipid in the cell membrane.
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> 3rd qns) alcohol. only DNA is not sol in alsohol..so what is left in the
> liquid will be protein..carbohydrate..lipid molecules.
>
> its so diff from e answers in e chemical forum...=( how?? savoy can help me?
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1) Sodium acetate salts are added to the cell extract in order to precipiate out the proteins, lipids, and other cell debris.
2) The detergent essentially disolves the plasma membrane of the cell, lysing the cell.
3) The alcohol will precipiate the DNA from solution. The main purpose of this step is not to remove residual proteins lipids, and carbohydrates (most of these are gone by this time), but rather to isolate the DNA from the salts used to precipitate out the proteins, lipids, and carbohydrates.
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Lily,
sorry about the late reply. I'd go with Yggdrasil's last response which is pretty much what your friends have given you.
Extracting DNA doesn't have to be difficult, it can be done with shampoo and precipitated out with ice-cold alcohol. It is the purification of DNA(getting rid of unwanted proteins & other macromolecules) that may at times be more difficult.
savoy
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Trust me: plasmid DNA, in particular, is subject to density gradient ultracentrifugation through ceasium chloride.
But it takes quite a lot of EtBr to do this type of purification. Thus, most lab-worker prefere non-EtBr extractions (which are faster as well).
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Hi Lily,
I found this on the web:
NaCl is to keep the DNA in its double helix form, otherwise it would denature and become "ssDNA".
I found this in the book "Biology Projects for Young Scientists"
"The salt contains positive charges that will neutralize the negative charges on the DNA extracted. If the +ve charges are not neutralized, they would repel one another and break down the DNA"
The detergent breaks down the lipids and makes the membranes dissolve.
Hope this helps.
I am new to this too and I am trying to understand each step as well.
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yep u r absolutely right! chemical properties of dna is negatively charged,for them to clump together,one has to add salt water which contain sodium cations to neutralise the dna molecules.
e rest is quite straight forward....
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Why, exactly, would meat tenderizer be used? I have seen some procedures on the web that call for this. They say something about removing proteins that the DNA is attached to. Would using Na acetate have the same effect?
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I think meat tenderizer is indeed used to degrade proteins that the DNA is bound to, and possibly to degrade cellular membrane proteins too. Both kiwi fruit and pineapples contain a similar enzyme (why we put pineapple on gammon steak) and DNA can be more easily extracted from them.
There are many different ways to extract DNA depending on what your getting it from and you want in terms of purity etc. For someone wanting to extract DNA at home the question is why they want to do this. If its just for some fun and to impress the chicks (in case you can't tell im being sarcastic) then there are very simple recipes on line to extract DNA from kiwi, you can actually see and pick up the long stringy molecule.
If your trying to extract your own DNA to clone yourself or build an army of supersoldiers or something then things get more precise and i'm not sure i'd like to help anyone out on this.
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If your trying to extract your own DNA to clone yourself or build an army of supersoldiers or something then things get more precise and i'm not sure i'd like to help anyone out on this.
I'm just trying to clone some of the pretty ladies. I don't have the patience to wait decades for them to grow up, so I am kind of stumped on that point. ;D
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I don't have the patience to wait decades for them to grow up
I don't think this will be a problem if you are a paedophile. LOL.
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I'm just trying to clone some of the pretty ladies. I don't have the patience to wait decades for them to grow up, so I am kind of stumped on that point.
Use hybridoma-like technology! Its fast! Believe me... ;) & I think u take face from actress A , lips of B, Legs of C, and [ i think , i should stop here...] ;)
& One confirmation that why the pineapple enzyme doesn't kill its own proteins is as the enzyme is in the vescicles and not free... BUt when we add soap it comes out, right?
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So anyone can help...what happen if water is added instead of alocohol and why?
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If you try to precipitate DNA with water instead of alcohol, it won't work. DNA is soluble in water.
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So anyone can help...what happen if water is added instead of alocohol and why?
in order to precipitate the DNA you need to get rid of the water that interacts with the DNA.
--> water thus has no effect (note that the phosphate backbone of DNA is negatively charged !)