Agarose Gel Electrophoresis, How It

Works and Its Uses

Article Published: February 10, 2022 | Karen Steward, PhD

Credit: iStock

Simple laws of physics dictate that when current is applied to a medium containing
charged species, those species will migrate towards the opposite charge.
Depending on the medium through which they are moving, other characteristics –
such as the size of the species present – can impact their movement, leading to
separation. This is the basis on which electrophoresis techniques, such as agarose
gel electrophoresis, are built – techniques that are widely used across the life
sciences.
 Contents

What is electrophoresis?

What is agarose gel electrophoresis?

How does gel electrophoresis work?

DNA gel electrophoresis steps, the gel electrophoresis machine,

electrophoresis buffer and electrical separation

How to read gel electrophoresis

What is the purpose of gel electrophoresis?

DNA agarose gel electrophoresis glossary

References

In this article, we will consider how agarose gel electrophoresis works, how it can
be interpreted and some of its purposes.

What is electrophoresis?

Electrophoresis is a technique that uses electrical current to separate DNA, RNA or

proteins based on their physical properties such as size and charge.

What is agarose gel electrophoresis?

Agarose gel electrophoresis is a form of electrophoresis used for the separation of

nucleic acid (DNA or RNA) fragments based on their size. Negatively charged
DNA/RNA migrates through the pores of an agarose gel towards the positively
charged end of the gel when an electrical current is applied, with smaller
fragments migrating faster. The resulting bands can then be visualized using
ultraviolet (UV) light.
RNA,1 however, tends to form secondary structures – sometimes multiple different
species for the same fragment – that affect the way it migrates. Consequently,
observed bands do not always represent their true sizes and images are blurry.
Native agarose gels (where conditions do not disrupt the natural structures of
analytes) therefore tend not to be used for the analysis of RNA sizes, although they
can give an estimate of quantity and integrity. Alternatives include northern
blotting and denaturing agarose gel electrophoresis2 that use conditions able to
disrupt secondary structures.

Agarose gels may also be used to separate proteins3 based on their size and
charge (unlike DNA/RNA, proteins vary in charge according to the amino acids
incorporated). However, due to the large pore sizes in agarose gels, proteins are
often separated on polyacrylamide gels that have smaller pores instead, offering
greater resolution for small protein molecules.

Continue reading below...

Article

Polyacrylamide Gel Electrophoresis, How It Works,

Technique Variants and Its Applications
Read More

We will therefore focus on DNA agarose gel electrophoresis for the remainder of
this article.

How does gel electrophoresis work?

Agarose is a component of agar. It forms a 3D gel matrix of helical agarose

molecules in supercoiled bundles held by hydrogen bonds, with channels and
pores through which molecules are able to pass. When heated, these hydrogen
bonds break, turning the agarose to liquid and allowing it to be poured into a mold
before it resets (Figure 1).

Figure 1: Pore formation and temperature-induced state transition in agarose gel.

Credit: Technology Networks.
The percentage of agarose included in a gel impacts the pore sizes and thus the
size of molecules that may pass through and speed at which they do so. The higher
the percentage of agarose, the smaller the pore size, thus the smaller the
molecules able to pass and the slower the migration. In the molecular biology lab,
0.7-1% agarose gel is typically used for day-to-day DNA separations, offering good,
clear differentiation of fragments in the range of 0.2-10 kb. Larger fragments may
be resolved using lower percentage gels, but they become very fragile and hard to
handle, while higher percentage gels will give better resolution of small fragments
but are brittle and may set unevenly.

What is gel electrophoresis used for?

Gel electrophoresis is used to separate mixtures of biomacromolecules, such

as DNA, RNA and proteins. This technique separates by molecular size and/or
charge. This is achieved by drawing molecules through a gel containing tiny
pores using an electrical field.

As DNA is not visible to the naked eye, an intercalating dye such as ethidium
bromide (EtBr) is incorporated in the gel during setting. This binds the DNA and
fluoresces under UV light, allowing the DNA fragments to be visualized. The more
DNA present, the brighter the band.

Samples mixed with a loading dye are placed in one end of the gel which is
immersed in running buffer. An electrical current is then passed through the gel by
electrodes at each end of the gel tank (Figure 2).
Figure 2: Illustration of an agarose gel electrophoresis setup. The agarose gel sits
in a tank of buffer, the samples mixed with loading dye are placed in wells at one
end of the gel and an electrical current is applied causing the negatively charged
DNA to move towards the positive electrode (anode [Updated, February 13, 2023]).
Credit: Technology Networks.

When the samples have run far enough to obtain sufficient separation, the gel is
removed from the tank and placed on a UV light box. The intercalating dye then
allows the sample bands to be visualized and their size determined in comparison
to a DNA ladder with known band sizes. The relationship between migration
distance and fragment size in non-linear, adding to the importance of including
size markers as a guide (Figure 3).
Figure 3: A) The illustration above depicts a typical result of DNA electrophoresis.
On the left, there is a size marker that is used as a reference for the length of the
sample DNA fragments (in base pairs). To the right of the marker are three
samples: Sample A, Sample B and Sample C. The image shows how smaller DNA
fragments move further through the agarose gel than the larger fragments of DNA.
B) The graph to the right of the image shows the nonlinear, relationship between
the size of the DNA fragments and the distance migrated. It is a negative curve,
and as DNA fragments get larger, they migrate less distance through the gel. Credit:
Mckenzielower, reproduced under the Creative Commons Attribution-Share Alike 4.0
International license.

DNA gel electrophoresis steps, the gel electrophoresis

machine, electrophoresis buffer and electrical separation

There are a number of key steps4 involved in choosing, setting up, running and
analyzing agarose gels that we will now consider.

1. Determine the required gel percentage – 0.7–1% agarose gel is typically

adequate for most applications, but it is important to choose a percentage
appropriate for your samples and expected fragment sizes. Combine the agarose
powder with the same buffer type to be used to run the gel and heat to melt the
mixture, avoiding boiling. Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE)
or tris-borate-EDTA (TBE)5 are often the buffers of choice, as tris-acid solutions are
effective buffers for slightly basic conditions, keeping DNA deprotonated and
soluble in water. The EDTA, a chelating agent, inactivates nucleases that may
damage the DNA being analyzed.

2. Pour a gel – Choose a gel casting mold and comb of the desired size, giving a
sufficient number of wells for all samples and ladders and well capacity to hold the
quantity of each sample to be loaded. Secure the open ends of the mold with a
casting frame or tape to contain the gel while it sets. Add DNA intercalating dye
into the bottom of the mold – for EtBr, 0.2-0.5 µg/ml is typically used. Evidence that
EtBr is a mutagen is still currently debated but consequently, many labs have
moved over to alternatives6 such as GelRed. Add the gel, careful not to overfill the
mold, and ensure the intercalating dye is evenly mixed. Don’t pour the gel when it’s
too hot or the mold may warp or break.

3. Mix samples/ladders with loading dye – Loading dyes perform multiple

functions. They allow the user to see where their otherwise colorless sample is,
making it easier to pipette the sample into the well accurately and thus reducing
the likelihood of cross-contamination of samples between the wells. When the gel
is running, the dye migrates with the sample, allowing the user to tell where in the
gel the sample has reached and prevent it from running too far and being lost into
the buffer. DNA samples without loading dye will also tend to disperse into the
running buffer when loaded as they are less dense. Most loading dyes therefore
contain glycerol or Ficoll which makes the sample-dye mix denser, so it settles in
the bottom of the wells. Bromophenol blue is a popular colorant choice, but some
also contain additional dyes such as xylene cyanol. While loading dyes can be
purchased, many labs choose to make their own.7

If you are running very small volumes of sample (e.g., less than 5 µl), it may be
advantageous to add a little water at this stage to make it easier to load the gel
wells effectively and evenly. Equally, if you expect the concentration of DNA in
some samples to be much higher than others, it may also be necessary to add
water to your sample-dye mix of these concentrated samples at this stage too. If
you do not, the strong signal given by these bands during visualization can mask
weaker bands or require the strong bands to be over exposed to view the weaker
ones, creating bright, distorted areas on the gel image.

4. Load the gel – Remove the casting frame/tape from the set gel and place it
in the gel tank, ensuring that the wells are at the negative end (black electrodes).
Fill the tank with running buffer (TAE or TBE) so that the gel is submerged. Carefully
remove the comb and gently pipette the sample-dye (and water if used) mix into
the wells. Try to avoid touching the edges of the wells with the pipette tip as they
may break and allow one sample to run into the next. Overloading wells can have
the same result. Large amounts of DNA can also slow down DNA migration during
running. Load marker ladders, preferably one at each end of the sample row.
Gels may not always run in a perfectly straight line so having a ladder at each end
makes it easier to determine the sizes of fragments present. A variety of ladders
are available with varying sizes indicated; chose one that is most appropriate for
the fragment sizes you expect to see.

5. Run the gel – Place the lid on the tank with the electrodes black to black and
red to red and plug the electrodes into a power pack, also black to black and red to
red. This, along with the gel tank, makes up the gel electrophoresis machine.
Make sure that the electrodes and lid are the correct way around otherwise your
samples will run backwards out of the wells and into the running buffer. Set the
time and voltage your gel will be run at; 120 V for 35 mins is a good approximation,
however this should be tailored to the gel percentage used and expected fragment
sizes being separated to give good electrical separation. Applying current to an
agarose gel will cause it to heat up, the higher the voltage the more it will heat so
when running low percentage gels, it is advisable to use lower voltages to prevent
melting. It can be tempting to increase the voltage to make a gel run faster.
However, this can result in “smiley gels”, where the bands are curved upwards at
each end, making it difficult to determine the correct band size. This is where the
gel has started to melt slightly making the bands run unevenly. This can also cause
bands to appear smeary and poorly defined.

6. Visualization – Once the samples have run most of the way down the gel
(the dye front will make this visible), turn off the power pack. Wearing gloves,
gently remove the gel in the mold from the tank, draining off excess running
buffer, and transfer to a UV box in an appropriate container for visualization.
Change gloves to prevent contamination of surrounding surfaces, door handles
etc. with intercalating dye from the gel or running buffer. If DNA fragments are
required for downstream applications, the corresponding bands can be carefully
excised from the gel with a scalpel blade while placed on a UV light box in a dark
room. Ensure you wear a UV face shield and keep skin covered while the light box
is on to prevent damage to your skin or eyes by the UV light.

Result visualization by Agarose Gel electrophoresis. Credit: Food and Agriculture Organization of the

United Nations via YouTube.

How to read gel electrophoresis

Agarose gels may be visualized on a UV light box in a dark room or using a self-
contained light box linked to a camera. Whichever system is utilized, UV light is
shone through the gel from below and bands of DNA fluoresce thanks to the
intercalating dye bound to them. This may be captured using a camera with a
specialized UV filter for your records. Marker ladders come with a guide to indicate
the size of each band they include. By comparing this to bands in sample lanes, the
sizes of the bands can therefore be determined. The relative amount of DNA
between samples may also be compared, as higher DNA concentrations will
produce brighter bands. An example is shown in Figure 4.
Figure 4: Agarose gel (2%) analysis of PCR-amplified products from DNA extracted
from a bronchoalveolar lavage (BAL) diagnostic specimen of a patient with
pulmonary symptoms. Credit: The Centers for Disease Control and Prevention.

What is the purpose of gel electrophoresis?

There are a number of reasons why the separation of DNA fragments may be
desirable, many of which are widely applicable across the life science disciplines.
Let’s consider some common purposes.

• Visualization of sample DNA

Separating and visualizing DNA fragments allows a user to determine:

Whether DNA is present in a sample – This can confirm, for example, if a

DNA extraction or a PCR has worked and in the context of a diagnostic test
can therefore determine if a sample is positive or negative.

The sizes of DNA fragments present – If performing a PCR or restriction

 digest, for example, is the band of the expected size? This can confirm if
genetic engineering experiments have been successful or may indicate the
presence or absence of genetic insertions, deletions8 or repeat regions9 that
can be used as a diagnostic tool for some genetic conditions. When preparing
DNA for next-generation sequencing, it is important that the fragmented DNA
for library preparation is of the correct size for efficient sequencing.

The amount of DNA present – Although there are more accurate methods
for precise DNA quantification,10 such as ultraviolet-visible spectroscopy,
when running samples on a gel, the intensity of the band produced can give a
rough idea of the amount of DNA in a sample relative to other samples.

How clean the sample is – While a smeared band may be expected in some
contexts, such as when running whole genomic DNA, a clear, crisp band may
generally be expected from a PCR or restriction digestion. Diffuse bands or
smears may indicate suboptimal PCR conditions or primers, incomplete
digestion or the presence of interfering contaminants such as RNA in the case
of a DNA sample.

• Separation of DNA fragments for purification

Where DNA fragments are required for downstream applications, such as
cloning,11 or following restriction digestion, it may be desirable to separate
out DNA fragments of a specific size from others in the total sample. To
achieve this, the digested or amplified sample may be run out on a gel and
the piece of gel containing the fragments of interest excised. Clean-up kits
and protocols12, 13 are available to purify the DNA from the agarose gel
before proceeding to downstream steps.

• Separation of DNA fragments for Southern blotting

Southern blotting is a technique used to detect specific DNA sequences in a
sample. In order to do that though, the DNA fragments first have to be
separated by agarose gel electrophoresis before they can be probed for
target sequences.
 • Electrophoretic mobility shift assays (EMSAs)
EMSA’s,14 also called gel shift assays, are used to detect interactions between
proteins and nucleic acids. Examples might include the binding of
transcription factors15 that promote or prevent gene expression. When a
protein binds to a fragment of DNA, it will alter the way it migrates through
an agarose gel, producing a “shift”. Therefore, by running different
combinations of DNA fragments with and without a putative DNA binding
protein, it is possible to determine when binding has or has not occurred and
thus determine the target sequence.

DNA agarose gel electrophoresis glossary

Term Definition

Agarose gel electrophoresis A form of electrophoresis used for the

separation of macromolecules, such as
DNA fragments, in an agarose matrix.

Chelating agent A chemical compound that reacts with

metal ions to form stable, water-soluble
metal complexes.

Denaturing agarose gels Agarose gels run under conditions that

disrupt the natural structure of DNA,
RNA or proteins, causing them to
unfold.

Deprotonation Removal of a proton (H+).

DNA ladder/marker ladder A solution of DNA fragments of known
sizes that can be used to extrapolate
the sizes of fragments in unknown
samples.

Electrical separation The separation of biomolecules, such as

DNA, RNA or proteins, according to
their size and/or charge using electrical
current.

Electrophoresis A technique that uses electrical current

to separate DNA, RNA or proteins
based on their physical properties such
as size and charge.

Electrophoretic mobility shift assays Also called gel shift assays, EMSAs are
(EMSA) an electrophoresis-based technique
used to detect interactions between
proteins and nucleic acids.

Gel electrophoresis machine Equipment used to perform gel

electrophoresis that normally consists
of a gel tank and power pack with
connecting electrodes.

Intercalating dye Dyes that bind double stranded DNA,

enabling them to be visualized under
UV light.

Loading dye A dye used to prepare DNA ladders and

samples for electrophoresis that
enables them to be seen with the naked
eye and increases their density to
prevent dispersion.
Mutagen A chemical or physical phenomenon
that promotes errors in DNA
replication.

Native agarose gels Agarose gels run under conditions that

allow the preservation of the natural
structure of biomolecules such as DNA,
RNA or proteins.

Northern blot A technique used to detect specific RNA

sequences in a sample that employs
electrophoresis for sample separation.

Nucleases Enzymes that cleave nucleic acids such

as DNA or RNA.

Polyacrylamide gel electrophoresis A form of electrophoresis used for the

separation of macromolecules, such as
nucleic acids and proteins, in a
polymerized acrylamide matrix.

Restriction digestion A process that uses enzymes to cut

DNA at specific sites according to the
surrounding DNA sequence.

Running buffer The buffer used to fill the tank in which

the gel is immersed and will be run. It
helps to maintain stable conditions.

Secondary structure The 3D structure adopted by a

polypeptide or polynucleotide resulting
from electrostatic attractions between
neighboring residues.
Southern blot A technique used to detect specific DNA
sequences in a sample that employs
electrophoresis for sample separation.

Transcription factors Proteins involved in the process of

converting, or transcribing, DNA into
RNA.

References
(Click to expand)

References

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Correction

The article erroneously stated that the positive electrode was the cathode, this was updated on February 13, 2023, to

correctly identify the anode as the positive electrode.

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