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Work of Salt, Isopropanol and Ethanol in DNA Extraction

  
  
  

Question:

What is the work of salt, isopropanol and ethanol in DNA extraction?

The Protein Man Says:

To identify bacteria and viruses in an environmental sample, diagnose disease pathologies, or examine a biological sample for forensic purposes, the DNA can be removed from the nucleus of a cell and its proteins can be separated by electrophoresis. Salt, isopropanol and ethanol are commonly used to precipitate DNA.                                                     

dna extraction, ethanol, isopropanol, DNA Extraction from Blood and Other Cell Tissue: Initial Phase  

The DNA extraction process begins with the mechanical separation of the nuclear contents from the rest of the cell, which is carried out by sonication, agitation and the addition of SDS detergents. To further break down cell components and then draw off the DNA associated proteins, researchers typically add ammonium, sodium acetate or similar salts during this stage of the procedure.  

DNA Extraction Using Ethanol Precipitation

Since DNA is insoluble in ethanol and isopropanol, the addition of alcohol, followed by centrifugation, will cause the DNA proteins to come out of the solution. When DNA concentration in the sample is heavy, the addition of ethanol will cause a white precipitate to form immediately. If the DNA concentration in the sample is low, isopropanol may work better than ethanol to precipitate the available proteins. In addition, isopropanol is often used for precipitating DNA from large volumes as less alcohol is used (see protocols below).

The ethanol and isopropanol can also wash away the remaining salt residue. After being washed in alcohol and subjected to a centrifuge, the precipitated DNA protein will form a pellet, which can be washed in alcohol again, dried, and re-suspended in a Tris or TE buffer. Be careful not to overdry the sample, since this can denature the DNA; just leave the washed pellet on the lab table for a few minutes. If isopropanol has been used during the extraction instead of ethanol, the sample may not adhere as tightly to the tube and may require a longer drying time.

General Protocol for Ethanol Precipitation

  1. Add 1/10 total volume of Sodium Acetate (3M, pH 5.2).

  2. Add 2-3X volume of at least 95% ethanol. Calculate after addition of sodium acetate.

  3. Incubate on ice for 15 minutes. In case of small DNA fragments or high dilutions overnight incubation gives best results.

  4. Centrifuge at >14,000 x g for 30 minutes at 4°C to prevent overheating the sample.

  5. Discard supernatant by decanting or pipetting, being careful not to throw out DNA pellet which may or may not be visible.

  6. Rinse with 70% Ethanol and centrifuge at >14,000 x g for 15 minutes at room temperature or 4°C.

  7. Discard supernatant, air dry pellet (5-20 minutes) and dissolve pellet in desired buffer.

  General Protocol for Isopropanol Precipitation

  1. Add 1/10 total volume of Sodium Acetate (3M, pH 5.2).

  2. Add 0.6-0.7X volume of room temperature isopropanol. Calculate after addition of sodium acetate. Room temperature isopropanol minimizes coprecipitation of salt.

  3. Immediately, centrifuge at >14,000 x g for 30 minutes at 4°C to prevent overheating the sample.

  4. Discard supernatant by decanting, being careful not to throw out DNA pellet which may or may not be visible. Isopropanol precipitated pellets are often difficult to see and loosely attached. Mark outside of tube before centrifugation for easy identification.

  5. Rinse with 70% Ethanol and centrifuge at >14,000 x g for 15 minutes at room temperature or 4°C.

  6. Discard supernatant, air dry pellet (5-20 minutes) and dissolve pellet in desired buffer.

Comments

Great informative article and thanks for sharing these information briefly. but one question is arise in my mind, In DNA precipitation, sometimes isopropanol is used, sometimes ethanol is used, what is the reason for using one not the other, and what's the rule when to use one not the other?
Posted @ Friday, April 05, 2013 4:51 AM by johnbartram
In DNA precipitation, sometimes isopropanol is used, sometimes ethanol is used, what is the reason for using one not the other, and what's the rule when to use one not the other? 
Isopropanol or Ethanol? 
Ethanol 
• DNA is more soluble in ethanol requiring a final concentration of ~75% ethanol and 0.5M salt. This means you would need to add 2-2.5 volumes of ethanol to your sample. 
• Ideal for short DNA pieces or very dilute samples 
• Storage at -20°C improves precipitation and also allows for long term storage. 
Isopropanol 
• DNA is less soluble in isopropanol requiring a final concentration of ~35% isopropanol and 0.5M salt. This means adding 0.7-1 volumes of isopropanol to your sample 
• Downside is salt is also less soluble and can co-precipitate. 
• Ideal for concentrated DNA samples 
• Storage at -20°C not recommended as salt precipitation can occur 
Which should I use? 
The main deciding factor is the volume of DNA sample you are precipitating. If you have room in the tube to add 2-2.5 volumes then use ethanol with a -20°C incubation for at least 1 hour, preferably overnight). IF you have a large volume and only have room for 1 volume then use isopropanol. This also allows for more rapid precipitation as incubations done at room temperature. Make sure to wash with 70% ethanol to remove salts! 
Posted @ Friday, April 05, 2013 9:32 AM by Colin Heath
my question is why 75% ethanol is added to wash the DNA pellet in the last steps. i mean we can put 100% ethanol also but why only 75%?
Posted @ Monday, May 26, 2014 1:22 PM by ayushi
Ayushi, 
The water in the 70% ethanol wash allows for an increase in solubility of the salts in the DNA pellet. This allows them to be removed from the DNA
Posted @ Tuesday, May 27, 2014 9:28 AM by Colin Heath
Use of 100% ethanol also causes complete removal of water from the DNA, which causes over-drying of DNA and eventually breakage(denaturation) of DNA molecules.
Posted @ Tuesday, May 27, 2014 10:48 AM by Raktim
this websit is informative and really give me more enlightment on DNA Extraction. It is lovely.i will be glad to have more update through my e-mail. 
thank
Posted @ Wednesday, July 23, 2014 1:13 PM by Salami abisola
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