As a follow-up to our article about 

ethanol precipitation of DNA and RNA, this

article explains the differences between DNA precipitation in ethanol and
isopropanol, helping you to figure out which method is the best choice for your
experiment.

Requirements for DNA Precipitation

First, let’s review the components we need to precipitate DNA or RNA with
ethanol:
1. Salt to neutralize the charge on the nucleic acid backbone. This causes the
DNA to become less hydrophilic and precipitate out of solution.
2. Ice to chill the sample. Lower temperatures promote the flocculation of the
nucleic acids so they form larger complexes that pellet under the centrifugal
forces of a microcentrifuge.
3. A nucleic acid concentration high enough to force the DNA out of solution (if
the concentration is not high enough, you can add a carrier nucleic acid or
glycogen to enhance the recovery).
4. A microcentrifuge to pellet the sample.

Isopropanol Vs. Ethanol: DNA Solubility

DNA is less soluble in isopropanol so it precipitates faster even at low
concentrations. [1] The downside however is that salt will also precipitate in
isopropanol. With ethanol, the DNA needs to be at a higher concentration to
flocculate but the salt tends to stay soluble, even at colder temperatures.
DNA precipitates in 35% isopropanol and 0.5 M salt. Using ethanol, the final
concentration needs to be around 75% with 0.5 M salt. So for the typical
precipitation protocol, isopropanol is added from between 0.7–1 volumes of
sample, and ethanol is added at 2-2.5 volumes of sample.

Choosing the Right Solvent: Sample Volume

If you are precipitating small volumes of DNA, and you can fit the required
amount of solvent into the sample tube, then ice-cold ethanol is the preferred
choice. You can chill it (in liquid nitrogen or at –80°C) to accelerate the
precipitation without the risk of precipitating excess salt. Afterwards, you need
to wash the pellet with 70% ethanol to remove any salt present.
Isopropanol is useful for large sample volumes e.g. the eluate you get after
using a large volume plasmid kit. Because less isopropanol is needed for
precipitation, you can often fit your sample and the solvent in one 15 ml tube.
However, because salts are generally less soluble in isopropanol than in
ethanol, they tend to co-precipitate with DNA. To minimize the likelihood of
salt precipitation, isopropanol precipitation is best at room temperature with
short incubation times.
Once you recover the DNA or RNA pellet from the isopropanol, wash it with
cold 70% ethanol to remove excess salt and to exchange the isopropanol for
ethanol. It is ok to chill the isopropanol-precipitated sample if you are sure the
sample doesn’t contain a lot of salt.
Because DNA is less soluble in isopropanol, isopropanol allows precipitation
of larger species and lower concentrations of nucleic acids than ethanol,
especially if you incubate at low temperatures for long periods of time.
If you do this, just remember to wash the pellet several times in 70% ethanol
after pelleting, to reduce the amount of salt you carry over.

In Short – Ethanol or Isopropanol?

Use Ethanol If:
1. You have the space to fit two volumes of ethanol to sample in your tube.
2. The sample needs to be stored for a long period of time and will be
chilled.
3. You need to precipitate very small DNA fragments.

Use Isopropanol If:

1. Your sample volume is large and you can only fit 1 volume of solvent
into your tube.
2. You need large molecular weight species.
3. The DNA concentration in your sample is low.
4. You are in a hurry and want to accelerate the precipitation of nucleic
acids at room temperature.

Handy Tips
 Ethanol precipitation of DNA:
 o Add 2 volumes of ethanol to the sample and freeze at –20°C for at
least 1 hour or overnight for best results.
o Centrifuge the sample at full speed for 20 minutes to collect all
material.
o Wash with 70% ethanol, then centrifuge for 10–15 minutes to
pellet the DNA.
o Remember to mark the side of the tube where the pellet is
expected to be and don’t let it out of your sight when decanting
the ethanol!

 Isopropanol precipitation of DNA:

o Avoid cold temperatures because of the excess salt precipitation
that can occur.
o To increase the yields precipitated, incubating the sample mixture
at room temperature for longer periods rather than chilling the
sample.
o When the DNA is pelleted, the pellet is sometimes more difficult to
see compared to the ethanol pellet. It can be clear and glassy.
Make sure, again, to note the side of the tube where the pellet
should be. Look for it before decanting the isopropanol and 70%
ethanol wash.
o After washing with ethanol, the pellet becomes visible and white.
Make sure it doesn’t slip off the side of the tube wall before
decanting the supernatant. Allow the tube to drain upside down
for a few minutes, let it air-dry, or use a centrifugal evaporator (5
minutes is enough) and then resuspend in buffer.

 Finally, for dry DNA pellets, heating the sample in buffer at 50–60°C will
help the DNA dissolve faster and won’t damage the DNA. Heating is also
suitable for RNA, in a water bath at temperatures not exceeding 42°C.
Over-dried DNA and RNA will take longer to dissolve so make sure not
to evaporate for too long.

So now you know the difference between ethanol and isopropanol

precipitation, and when to use each method. Good luck with your DNA
precipitations!

References
1. Green MR, Sambrook J. Precipitation of DNA with Isopropanol. Cold Spring
Harb Protoc. 2017(8):pdb.prot093385.
Originally published on December 10, 2009. Updated and republished in
August 2021.

Preparation of 1M Magnesium Chloride

(MgCl2) Stock Solution
 ADMIN    LEAVE A COMMENTON PREPARATION OF 1M MAGNESIUM CHLORIDE (MGCL2) STOCK SOLUTION

Overview:
Magnesium Chloride (MgCl2) is soluble in water. Magnesium chloride is available
in anhydrous as well as hydrated forms. Since anhydrous Magnesium chloride is
hygroscopic (quickly absorbs water from the air), it is convenient to use hydrated
forms that are easy to weigh out the right amount accurately. For this reason,
Magnesium chloride hexahydrate (MgCl2.6H2O, Molecular Weight: 203.30) is
often used to prepare a 1 M Magnesium chloride solution.
A 1M stock solution can be prepared by dissolving 203.3 g of  MgCl2.6H2O
(Molecular Weight: 203.30) in water to a final volume of 100 ml. 
In the following section, we describe a procedure to prepare 100 ml of 1M
MgCl2 stock solution.
REQUIREMENTS
Reagents and solutions
Magnesium chloride (MgCl2.6H2O) (Molecular Weight: 203.3)
Deionized / Distilled water
Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer (optional)

Composition
1 M Magnesium chloride
Objective
Preparation of 100 ml 1 M Magnesium chloride
PROCEDURE
Step 1: To prepare 100 ml of 1 M Magnesium chloride, weigh out 20.33 g
MgCl2.6H2O (Molecular Weight: 203.30) in a 250 ml conical flask/beaker. Add
800 ml deionized/Milli-Q water and mix it. 
Tip:
One can use manual shaking using a glass pipette to mix the ingredients. Magnetic
stirrer makes the dissolving process easy and convenient.
Precaution:
Do not dissolve in 100 ml of deionized / Milli-Q water. In most cases, solution
volume increases when a large amount of solute is dissolved in a solvent.
Step 2: Adjust the volume to 100 ml with deionized / Milli-Q water. Mix it
again.
Step 3: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi)
from 121-124°C on liquid cycle).
Note:
The solution can also be sterilized by filtering through a filter unit containing a
membrane with a pore size of 0.22 µm or less under sterile conditions.

Storage
The solution can be stored in the cold room.

Follow the table to prepare Magnesium chloride of various concentrations and volumes (10,
ml, 500 ml, 1,000 ml, and 10,000 ml).

Solution
concentration / Volume 10 ml 100 ml 500 ml 1000 ml

0.1 M 0.2033 g 2.033 g 10.165 g 20.33 g

10.165
0.5 M 1.0165 g g 50.825 g 101.65 g Required
amounts of
1M 2.033 g 20.33 g 101.65 g 203.30 g MgCl2.6H2O

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A Quick Guide on DNA Precipitation and
Protocol
DNA Extraction, Genetic Education / By Dr Tushar Chauhan / 17/04/2019 / 9 minutes of reading
“DNA precipitation is a process of nucleic acid (DNA/ RNA) precipitation using alcohol and
salt. Ethanol and isopropanol are the two most common types of alcohol used in the DNA
precipitation protocol.”
An important step of the DNA extraction protocol is DNA precipitation. DNA looks like cotton
threads when precipitated. However, read-to-use DNA kits, used commonly in recent times,
don’t need a precipitation step. It directly dissolves DNA in the elution buffer.  

Traditional DNA extraction protocols rely on the precipitation step, it’s a kind of validation step,
that indicates the presence of DNA. And that’s why scientists use this method so often. 

Besides the precipitated DNA has lucrative applications too which we will discuss in this article.
Alcohol is a common DNA precipitation agent, two common alcohol used in DNA extraction are
isopropanol and ethanol. 

 A few common steps in the traditional DNA extraction protocol are

 Lysis of cell wall or cell membrane

 Lysis of nuclear membrane
 Isolation of nucleic acid
 Precipitation of DNA
 Washing of DNA
 Dissolving DNA
Related article:
Different Types of DNA Extraction Methods

Take a look at the figure below,

 A brief outline of the DNA
extraction steps.
DNA precipitation is an amazing thing, it’s the only reaction in which we can visualize DNA by
eyes, not like the spiral or helical structure explained in the literature.  
 If you are a student then it’s important for you to understand the process of how lysis,
precipitation and elution occur during the process of DNA isolation. It will boost your DNA
extraction knowledge trust me. 

So in this article, I will explain to you the process of DNA precipitation and will give you a
common DNA precipitation protocol that indeed helps you.  

Stay tuned. 

Key Topics:
 What is DNA precipitation?
o Definition: 
o Mechanism of DNA precipitation:
 Importance of DNA precipitation: 
 Chemicals used in the DNA precipitation:
o Alcohol used in the DNA precipitation:
 A common DNA precipitation protocol:
 Conclusion:

What is DNA precipitation?

Alcohol precipitation precipitate or aggregate DNA into the visible cotton thread-like substance
is known as the process of DNA precipitation

Definition: 

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

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

The water has a partial positive charge and a partial negative charge. The positive charge of the
water molecule interacts with the negative charge of the DNA (the PO3-) and dissolves it.
However, the interaction is not so strong.
The image shows the intermolecular interaction between water and DNA.
The salt and alcohol make the DNA more water hydrophobic. Once we add the salt into the DNA
solution, the negative charge of the DNA interacts with the positive charge of the salt, instead of
the positive charge of the water.

It’s possible for DNA to bind with water but because of the “lower dielectric constant alcohol”,
the complex of DNA and salt is protected by an alcohol shield. And henceforth it can’t react with
water. 

This chemical reaction makes it possible to visualize DNA like a cotton thread and aggregates as
a precipitate. For a more in-depth explanation of the mechanism of the DNA precipitation please
read our previous article:  Role of alcohol in DNA extraction.

In addition to this, the alcohol also does another job to increase the efficiency and yield of DNA
precipitation. Instead of the negative charge of the DNA, the water molecule remains busy
interacting with the negative charge of the alcohol. 

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 precisely the nucleic acid) remains dissolved in the water.

(polar solution dissolve in polar and nonpolar solution dissolve in nonpolar solution)
Here, the interaction affinity is strong between the H+ and P03– of the water and DNA,
respectively, hence both interact with each other. This interaction is necessary to separate DNA
even into the next level of structure that is 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), the final centrifugation step will separate DNA and remove
impurities as well. 

I hope you will understand what I mean to say.

Conclusively, precipitation separates DNA from other macromolecules present in the solution. 

Now come to the point.

What does 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 is not shielded, the precipitates will not
form.

The higher dielectric constant of water repels interactions 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 of
salts and negative charge of the DNA interacts with one another and we can get more precipitate.

Take a look at the pictorial representation here,

Read more articles related to this topic,

1. CTAB DNA extraction buffer for plant DNA extraction

2. Proteinase K DNA extraction method
3. Phenol chloroform DNA extraction method
Importance of DNA precipitation: 
DNA precipitation is used to precipitate DNA into a visible solid form. 

It removes impurities from the DNA. 

It can also be used to store DNA in dried form, precipitated DNA can be stored for a longer
period of time. 

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 advise using sodium acetate.

Alcohol used in the DNA precipitation:

Alcohol is a key ingredient in not only DNA precipitation but also in DNA washing and DNA
storing. Two commonly used alcohols are isopropanol and ethanol.

You may wonder why not methanol or other alcohol? Why only ethanol or isopropanol is used? 

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 the dielectric
constant is 33.

The boiling point of methanol is lower than the ethanol and isopropanol while the dielectric
constant is higher. The lower the dielectric point, the higher the precipitation efficiency. We have
already discussed why.

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

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

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

Use 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, which 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.

Hypothetical graph of dielectric constant vs the rate of DNA precipitation using different
alcohol.
A common DNA precipitation protocol:
DNA precipitation is a subsidiary step in the DNA extraction process, when performed correctly
will always give the best purity and yield of DNA. 

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 DNA precipitates appear (see the figure below) centrifuge the precipitated DNA at
10,000 rpm for 2 minutes.
 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 it several times.
 Centrifuge the sample at 10,000 rpm for 2 minutes.
 Remove the alcohol and air dry the pellets.
 Once the pellets dry properly, dissolve them in the TE buffer at a pH of nearly 8.2.

Pictorial representation of the process of DNA precipitation.

Why is chilling 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 temperatures, high “flok” or aggregates are obtained by the addition of some of the
precipitating agents.
The addition of chilled ethanol facilitates higher flocculation in the process viz the more DNA
precipitation.

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.

Pro-tips:
For low DNA concentrate use 10 to 20 micrograms of glycogen and incubate the sample
overnight at low temperature to increase the yield of DNA precipitate.

or

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

Both modifications greatly increase the yield of DNA precipitation for smaller fragment nucleic
acid or low concentrate nucleic acid.

Notedly a double quantity of ethanol is required to precipitate the DNA.

 Precipitated
DNA from our lab.
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 minutes.
 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 it several times.
 Centrifuge the sample at 10,000 rpm for 2 minutes.
 Remove the alcohol and air dry the pellets.
 Once the pellets dry properly, dissolve them in the TE buffer at a pH of nearly 8.2.
 If not dissolved properly, heat the sample at 60°C until it dissolves.
 Image credit:
www.sciencelearn.org.nz
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.

Conclusion:
The use of manual or traditional DNA extraction protocol is quite uncommon, nowadays;
however, the manual method saves cost. 

Precipitation allows us to visualize DNA, it is a process, like magic; a moment ago you see
nothing but when you shake it you can see DNA. That’s an amazing experience, trust me. 

If you are really interested in pursuing your career in genetics, you should learn these things.
Achieving highly pure DNA using manual methods is an art. Timely, when you do this
repeatedly you will get expertise in it. 

I highly recommend you to go through this technique, do it yourself, it will enhance your
knowledge and skills.

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