“DNA precipitation is a process in which the nucleic acid is precipitated using alcohol and salt. Ethanol and isopropanol are the two most common types of alcohol used in the DNA precipitation protocol.”

DNA precipitation is an important step in the DNA extraction protocols, we can say especially, for traditional DNA extraction protocols.

DNA extraction protocols nowadays are robust and extraordinary uses such chemistry that directly dissolve DNA.

The traditional DNA extraction protocol completed in the following steps:

Lysis of cell wall or cell membrane

Lysis of nuclear membrane

Isolation of nucleic acid

Precipitation of DNA

Washing of DNA

Dissolving DNA

We had covered enough article on DNA extraction. Here we will directly discuss the “DNA precipitation” step.

Let’s start the discussion on DNA precipitation.



What is DNA precipitation?

DNA precipitation is a process in which the DNA is precipitated or aggregated into the visible cotton thread like precipitates using alcohol and salt.

The DNA precipitation also removes the impurities from DNA.

“The precipitation is a process in which the reaction between solute and solvent creates insoluble aggregate called precipitate and the process is called precipitation.”

The DNA is hydrophilic in nature which dissolves in water but not in the alcohol.

The water has a partial positive charge and a partial negative charge. The positive charge of the water molecule interact with the negative charge of the DNA (the PO3-) and dissolve it. However, the interaction is not so strong.

The salt and alcohol make the DNA more water hydrophobic, once we add the salt into the DNA, the negative charge of the DNA interact with the positive charge of the salt, instead of the positive charge of the water.

Read our amazing article on DNA: DNA story: The structure and function of DNA

It might bind to the water but thanks to the alcohol, the “lower dielectric constant alcohol” protect the salt and DNA complex by shielding it against the water.

The DNA is visible like a cotton thread and aggregate as a precipitate due to this chemical reaction.

For a more in-depth explanation on the mechanism of the DNA precipitation please read our previous article: Role of alcohol in DNA extraction.

Furthermore, instead of the negative charge of the DNA, the water remains busy in interaction with the negative charge of the alcohol that also increase the precipitation efficiency and the overall yield of the DNA precipitate.

The entire mechanism of precipitation is based on the polarity of the solution and the dielectric constant of the solvent.

Mechanism of DNA precipitation:

The DNA is a polar solution and so the water is. Therefore, by intermolecular interaction, the DNA (more preciously the nucleic acid) remains dissolved in the water.

(polar solution dissolve in polar and non-polar solution dissolve in non-polar solution)

Here, the interaction affinity is strong between the H+ and P0 3 – of the water and DNA, respectively, hence both interact with each other.

The entire solution contains not only the DNA but some of the other impurities too. Thanks to this, it is necessary to separate DNA even into the next level of structure that visibly separable.

A precipitate is a visible form of DNA (Although it does not look like the spiral DNA, it is just a clump of white aggregates), by doing centrifugation we can separate it from other impurities. And this is the only purpose of the precipitation because immediately after the precipitation and washing we have to dissolve it once again in the polar solution.

I hope you will understand what I mean to say.

The precipitation process is just a modification in the DNA extraction protocol that facilitates separation of DNA from other macromolecules.

Now come to the point.

What actually, the alcohol do?

Here not only the alcohol but some of the salts such as sodium acetate, ammonium acetate, sodium chloride and lithium chloride perform a significant role.

The alcohol guards the salt – DNA interaction, if it not shield, the precipitates will not form.

The higher dielectric constant water repels or hinders in the interaction of positive ions of the salt and negative ions of the DNA so it is very difficult to dissolve DNA only using salt.

By adding the alcohol such as ethanol and isopropanol, the lower dielectric constant of the alcohol protects the complex from water, and as we shake more the tube more positive charge and negative charge of the DNA interacts with one another and we can get more precipitate.

Read more articles related to this topic,

Chemicals used in the DNA precipitation:

We had already given the name of all the chemicals used in the DNA precipitation.

Some of the common salts used in the DNA precipitation are,

Sodium acetate = 0.3 to 2M

Sodium chloride = 0.2 to 0.3M

Ammonium acetate = up to 5M

Lithium chloride = 0.10M

Lithium chloride is the best choice for RNA precipitation but it is as good as sodium acetate for DNA precipitation.

Ammonium acetate is one of the choices but not recommended for all types of DNA samples.

Sodium chloride and sodium acetate are the two best choices for DNA precipitation in which I personally more favours the sodium acetate.

Alcohol used in the DNA precipitation:

Alcohol is a key factor in not only the DNA precipitation but also in DNA washing and DNA storing.

Ethanol and isopropanol are commonly used in DNA precipitation.

But the question arises in mind about methanol and other alcohol, why to use only ethanol and isopropanol and not methanol?

Why methanol and other alcohol are not used in DNA precipitation?

Well, the DNA precipitation mechanism is solely dependent on the dielectric constant of the solvent.

Let’s go through some of the numbers:

Solvent Boiling point (°C) Dielectric constant Water 100 80 Methanol 68 33 Ethanol 78 ~24.3 Isopropanol 98 ~20.1

Take a close look at the methanol, the boiling point of methanol is 68°C and dielectric constant is 33.

The boiling point of methanol is lower than the ethanol and isopropanol while the dielectric constant is higher.

Lower the dielectric point, higher the precipitation efficiency. We already discuss it why.

Furthermore, Boiling point of both the champions is higher that means it is safer to use.

In addition to this, the methanol is toxic to us, therefore, the Isopropanol and the ethanol are the two best choices for the DNA precipitation.

However, if needed we can use methanol, it is totally fine and works nicely (not good for plasmid DNA extraction).

Use directly the 95% alcohol in DNA precipitation.

So the conclusion of the story is, use isopropanol, it precipitates best. But wait, is there any problem with it?

Practically, the isopropanol also precipitates the salt too, means that impurities still remain in our DNA and as we knew that the salts are the most dangerous inhibitors in the PCR reaction.

So, using ethanol is a wise decision for our DNA precipitation.

A common DNA precipitation protocol:

The DNA precipitation is a subpart of any DNA extraction methods, yet it is very crucial to do it properly.

Let us start our protocol using two of the best chemicals for DNA precipitation, ethanol and sodium acetate.

Prepare the sodium acetate solution of 2M at pH 5.2.

Add the 1/10 volume of sodium acetate to the nucleic acid lysate solution.

Add a doubled volume of pre-chilled ethanol.

Invert the tubes several times gently to precipitate the DNA.

After the DNA precipitates appear (see the figure below) centrifuge the precipitated DNA at 10,000 rpm for 2 munites.

Remove the supernatant carefully without disturbing the pellets.

Dry the pellets with tissue paper or air dry.

Now add 70% alcohol and wash the sample by inverting several times.

Centrifuge the sample as 10,000 rpm for 2 minutes.

Remove the alcohol and air dry the pellets.

Once the pellets dry properly, dissolve it in the TE buffer at pH nearly 8.2.

Why chilling is necessary for DNA precipitation?

The process of flocculation is very important for obtaining a higher yield of the precipitate. The flocculation is achieved by chilling the sample.

At lower temperature, high “flok” or aggregates are obtained by addition of some of the precipitation agents.

Addition of chilled ethanol facilitates higher flocculation in the process viz the higher precipitation of DNA.

In some other protocols, if precipitates are not achieved, the sample is incubated at the lower temperature (-20°C) or in the liquid nitrogen for some time to achieve precipitation.

It is a very good practice for plasmid DNA or bacterial DNA precipitation.

Tips: For low DNA concentrate use 10 to 20 μg of glycogen and incubate the sample overnight at low temperature increases the yield of DNA precipitation.

or

Add 10mM of MgCl2 to the precipitate and incubate for 1 to 1.5 hours at -20°C before centrifugation.

Both the modification greatly increases the yield of DNA precipitation of smaller fragment nucleic acid or low concentrate nucleic acid.

For plasmid DNA or low concentrate DNA use this DNA precipitation protocol:

Prepare the sodium acetate solution of 0.5M at pH 5.2.

Add the 1/10 volume of sodium acetate to the nucleic acid lysate solution.

Add a doubled volume of pre-chilled ethanol.

Invert the tubes several times gently to precipitate the DNA.

Add 10mM of MgCl2 and incubate at -20°C for 1 hour.

Centrifuge the DNA precipitated DNA at 10,000 rpm for 2 munites.

Remove the supernatant carefully without disturbing the pellets.

Dry the pellets with tissue paper or air dry.

Now add 70% alcohol and wash the sample by inverting several times.

Centrifuge the sample as 10,000 rpm for 2 minutes.

Remove the alcohol and air dry the pellets.

Once the pellets dry properly, dissolve it in the TE buffer at pH nearly 8.2.

If not dissolve properly, heat the sample at 60°C until it dissolved.

Tips: When you are using the isopropanol, do not freeze the sample or do not follow the chilling method because it precipitates more salts along with DNA.

The doubled quantity of ethanol is required to precipitate the DNA.

Conclusion:

The world of DNA extraction is ruled by ready to use DNA extraction kits nowadays. However, extracting DNA manually and visualizing the process of how DNA precipitate forms, is an amazing experience for anyone.

Everyone should learn the manual methods of DNA extraction. Achieving pure DNA with good quantity is an art. Learn manual things, it will enhance your knowledge and expertise.