DNA Extraction

[Bioc]

Hi, I saw this type of experiment on the net, and I don't get a few points. I'm talking about the experiment where you lyse a sample (peas, meat, etc) with a blender, then add detergent and salty water, and finally add alcohol to the mix to precipitate the DNA.

 

What I don't quite get about this experiment is how can alcohol (ethanol) and water form two phases, since ethanol is soluble in water. I think maybe is because the water molecules are solvating the salt and the detergent. Also, in the case of plant cells, I'm not really sure how can detergent break the cell walls. Last, how can salt help precipitate DNA? I know that DNA is negatively charged, it occurs to me that it has a higher affinity to Na+ ions than Cl-.

[CharonY]

Ethanol is used to precipitate DNA out of solution (i.e. you add several volumes of high-percentage ethanol to your sample). So you only have one phase that has a high (final) percentage of ethanol, which results in DNA to precipitate. After centrifugation the pellet is usually washed additionally in 70% EtOH to remove salts. Salts are indeed used as counterions to facilitate DNA precipitation (usually sodium acetate). The excess of Na+ is used to counterbalance the phosphate group.

 

 

You only have two phases if you use phenol extraction to remove proteins (a common step during nucleic acid extraction).

Detergents do not affect cell walls much. Additional disruption methods (as e.g. bead beating) may be necessary.

 

[Bioc]

Mmmm, a single phase heavy on ethanol makes more sense to me. I saw in a webpage that if you pour cold ethanol slowly, then two phases can form, but that wouldn't explain why would the DNA and the Na+ ions diffuse to the alcohol layer and form the precipitate. I am talking about the simplest form of the experiment btw, the one you can do in your house . None of the websites I have seen mention the use of something to break the wall, so detergent must be enough to atleast cause leakage of the cell material.

 

This is one of the websites I checked:

 

http://learn.genetics.utah.edu/content/labs/extraction/howto/