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Topic: The extraction of DNA  (Read 19998 times)

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gera19

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The extraction of DNA
« on: February 21, 2006, 05:48:37 AM »
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?
« Last Edit: February 21, 2006, 05:59:32 AM by lily »

Offline Albert

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Re:The extraction of DNA
« Reply #1 on: February 21, 2006, 06:12:28 AM »
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.

gera19

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Re:The extraction of DNA
« Reply #2 on: February 22, 2006, 04:11:49 AM »
for the 3rd question, is the answer liquid soap molecules(greasy parts) and proteins?

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Re:The extraction of DNA
« Reply #3 on: February 22, 2006, 06:38:18 AM »
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).
« Last Edit: February 22, 2006, 08:00:57 AM by Albert »

Offline Donaldson Tan

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Re:The extraction of DNA
« Reply #4 on: February 23, 2006, 07:40:38 PM »
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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Re:The extraction of DNA
« Reply #5 on: February 24, 2006, 06:12:37 AM »
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.
« Last Edit: February 24, 2006, 08:52:42 AM by Albert »

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Re:The extraction of DNA
« Reply #6 on: February 24, 2006, 08:48:39 AM »
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.
« Last Edit: February 24, 2006, 09:39:45 AM by geodome »
"Say you're in a [chemical] plant and there's a snake on the floor. What are you going to do? Call a consultant? Get a meeting together to talk about which color is the snake? Employees should do one thing: walk over there and you step on the friggin� snake." - Jean-Pierre Garnier, CEO of Glaxosmithkline, June 2006

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Re:The extraction of DNA
« Reply #7 on: February 24, 2006, 01:08:27 PM »
Ok, here is the procedure I've followed so far.

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Re:The extraction of DNA
« Reply #8 on: February 24, 2006, 05:12:51 PM »
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.

gera19

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Re:The extraction of DNA
« Reply #9 on: February 24, 2006, 11:37:49 PM »
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..
>
> 2nd qns) soap. soap will destroy the plasma membrane of the cell. it breaks
> down the lipid in the cell membrane.
>
> 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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Re:The extraction of DNA
« Reply #10 on: February 25, 2006, 01:39:46 AM »
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.

savoy7

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Re:The extraction of DNA
« Reply #11 on: February 26, 2006, 12:50:18 AM »
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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Re:The extraction of DNA
« Reply #12 on: February 27, 2006, 09:10:23 AM »
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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lawrence

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Re:The extraction of DNA
« Reply #13 on: March 02, 2006, 03:21:19 PM »
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.


gera19

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Re:The extraction of DNA
« Reply #14 on: March 06, 2006, 08:36:36 AM »
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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