Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA),

ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix.[1]
DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification
of DNA via PCR, but may be used as a preparative technique prior to use of other methods such
as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further
characterization.
Separation
The term "gel" in this instance refers to the matrix used to contain, then separate the target
molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is
chosen based on the specific weight and composition of the target to be analyzed. When
separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually
composed of different concentrations of acrylamide and a cross-linker, producing different sized
mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few
hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet
porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled
using appropriate safety precautions to avoid poisoning. Agarose is composed of long
unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large
pores allowing for the separation of macromolecules and macromolecular complexes.
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules
through the gel matrix. By placing the molecules in wells in the gel and applying an electric
field, the molecules will move through the matrix at different rates, determined largely by their
mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively
charged or toward the cathode if positively charged.[2]
In simple terms: Electrophoresis is a procedure which enables the sorting of molecules based on
size and charge. Using an electric field, molecules (such as DNA) can be made to move through
a gel made of agar. The molecules being sorted move through the space in gel material. The gel
is placed in an electrophoresis chamber, which is then connected to a power source. When the
electric current is applied, the larger molecules move more slowly through the gel while the
smaller molecules move faster. The different sized molecules form distinct bands on the gel.
Visualization
Gel electrophoresis
After the electrophoresis is complete, the molecules in the gel can be stained to make them
visible. Ethidium bromide, silver, or Coomassie Brilliant Blue dye may be used for this process.
Other methods may also be used to visualize the separation of the mixture's components on the
gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the
gel under ultraviolet lighting conditions, often using a Gel Doc. If the molecules to be separated
contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.
If several samples have been loaded into adjacent wells in the gel, they will run parallel in
individual lanes. Depending on the number of different molecules, each lane shows separation of
the components from the original mixture as one or more distinct bands, one band per
component. Incomplete separation of the components can lead to overlapping bands, or to
indistinguishable smears representing multiple unresolved components.
Bands in different lanes that end up at the same distance from the top contain molecules that
passed through the gel with the same speed, which usually means they are approximately the
same size. There are molecular weight size markers available that contain a mixture of molecules
of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples,
the bands observed can be compared to those of the unknown in order to determine their size.
The distance a band travels is approximately inversely proportional to the logarithm of the size
of the molecule.
Applications
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and
biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and
a gel imaging device. The image is recorded with a computer operated camera, and the intensity
of the band or spot of interest is measured and compared against standard or markers loaded on
the same gel. The measurement and analysis are mostly done with specialized software.
Depending on the type of analysis being performed, other techniques are often implemented in
conjunction with the results of gel electrophoresis, providing a wide range of field-specific
applications.
Nucleic acids
In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is
due to the naturally-occurring negative charge carried by their sugar-phosphate backbone.[3]
Double-stranded DNA fragments naturally behave as long rods, so their migration through the
gel is relative to their size or, for cyclic fragments, their radius of gyration. Single-stranded DNA
or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a
complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen
bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause
them to behave as long rods again.[4]
Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the
"Chain termination method" page for an example of a polyacrylamide DNA sequencing gel.
Characterization through ligand interaction of nucleic acids or fragments may be performed by
mobility shift affinity electrophoresis.
Electrophoresis of RNA samples can be used to check for genomic DNA contamination and also
for RNA degradation. RNA from eukaryotic organisms shows distinct bands of 28s and 18s
rRNA, the 28s band being approximately twice as intense as the 18s band. Degraded RNA has
less sharpely defined bands, has a smeared appearance, and intensity ratio is
less than 2:1.
Proteins

SDS-PAGE autoradiography - The indicated proteins are present in different

concentrations in the two samples.
Proteins, unlike nucleic acids, can have varying charges and complex shapes,
therefore they may not migrate into the polyacrylamide gel at similar rates, or
at all, when placing a negative to positive EMF on the sample. Proteins
therefore, are usually denatured in the presence of a detergent such as sodium dodecyl
sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge.[1]
Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per
gram of protein), so that the resulting denatured proteins have an overall negative charge, and all
the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods
instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins
migrate in the gel is relative only to its size and not its charge or shape.[1]
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous
polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.
Characterization through ligand interaction may be performed by electroblotting or by affinity
electrophoresis in agarose or by capillary electrophoresis as for estimation of binding constants
and determination of structural features like glycan content through lectin binding.
Agarose gel electrophoresis
is a method used in biochemistry and molecular biology to separate a mixed population of DNA and RNA
fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.[1]
Nucleic acid molecules are separated by applying an electric field to move the negatively charged
molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer
ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is
called sieving.[2] Proteins are separated by charge in agarose because the pores of the gel are too large
to sieve proteins.
Applications
 Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in
restriction mapping of cloned DNA.
 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to
Northern transfer.
Agarose gels are easily cast and handled compared to other matrices and nucleic acids are not
chemically altered during electrophoresis. Samples are also easily recovered. After the
experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.
How to make 1% agarose gel: In a small beaker, mix 32mL of purified H2O, 4mL of 10x TBE
buffer or 10x whichever buffer going to be used in electrophoresis, and 0.4g of agarose powder.
Heat on hot plate, or microwave, until solution is almost boiling (about 98 degrees Celsius).
Immediately pour solution into electrophoresis mold with notch (well) comb intact, let settle for
approximately 30 minutes, or until it appears firm. Be careful, a gel which is perfectly molded is
still extremely fragile. Remove notch (well) comb carefully, but quickly. Wrap in plastic and
place in refrigerator if gel is not for immediate use.
There are limits to electrophoretic techniques. Since passing current through a gel causes
heating, gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to
reduce pH changes due to the electric field, which is important because the charge of DNA and
RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution.
Further, different preparations of genetic material may not migrate consistently with each other,
for morphological or other reasons.
Factors affecting migration of nucleic acids
The most important factor is the length of the DNA molecule, smaller molecules travel faster.
But conformation of the DNA molecule is also a factor. To avoid this problem linear molecules
are usually separated, usually DNA fragments from a restriction digest, linear DNA PCR
products, or RNAs.
Increasing the agarose concentration of a gel reduces the migration speed and enables separation
of smaller DNA molecules. The higher the voltage, the faster the DNA moves. But voltage is
limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease
the resolution (above about 5 to 8 V/cm).
Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with
different speeds (slowest to fastest: nicked or open circular, linearised, or supercoiled plasmid).
Visualization: ethidium bromide (EtBr) and dyes
The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis
is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated
into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV
light, any band containing more than ~20 ng DNA becomes distinctly visible. EtBr is a known
mutagen, however, so safer alternatives are available.
Even short exposure of nucleic acids to UV light causes significant damage to the sample. UV
damage to the sample will reduce the efficiency of subsequent manipulation of the sample, such
as ligation and cloning. If the DNA is to be used after separation on the agarose gel, it is best to
avoid exposure to UV light by using a blue light excitation source such as the Xcitablue UV to
blue light conversion screen from Bio-Rad or Dark Reader from Clare Chemicals. A blue
excitable stain is required, such as one of the SYBR Green or GelGreen stains.
Blue light is also better for visualization since it is safer than UV (eye-protection is not such a
critical requirement) and passes through transparent plastic and glass. This means that the
staining will be brighter even if the excitation light goes through glass or plastic gel platforms.
SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more expensive, but 25
times more sensitive, and possibly safer than EtBr, though there is no data addressing its
mutagenicity or toxicity in humans.[3]
SYBR Safe is a variant of SYBR Green that has been shown to have low enough levels of
mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations.[4] It
has similar sensitivity levels to EtBr,[4] but, like SYBR Green, is significantly more expensive. In
countries where safe disposal of hazardous waste is mandatory, the costs of EtBr disposal can
easy outstrip the initial price difference, however.
Since EtBr stained DNA is not visible in natural light, scientists mix DNA with negatively
charged loading buffers before adding the mixture to the gel. Loading buffers are useful because
they are visible in natural light (as opposed to UV light for EtBr stained DNA), and they co-
sediment with DNA (meaning they move at the same speed as DNA of a certain length). Xylene
cyanol and Bromophenol blue are common loading buffers; they run about the same speed as
DNA fragments that are 5000 bp and 300 bp in length respectively, but the precise position
varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and
Orange G which run at about 125 bp and 50 bp.
Visualization can also be achieved by transferring DNA to a nitrocellulose membrane followed
by exposure to a hybridization probe. This process is termed southern blotting.
Percent agarose and resolution limits
Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50
base pair to several megabases (millions of bases) using specialized apparatus. The distance
between DNA bands of a given length is determined by the percent agarose in the gel. The
disadvantage of higher concentrations is the long run times (sometimes days). Instead high
percentage agarose gels should be run with a pulsed field electrophoresis (PFE), or field
inversion electrophoresis.
Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb
DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in
electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical
polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may
break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1%
gels are common for many applications.
Buffers
There are a number of buffers used for agarose electrophoresis. The most common being:
Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE) and lithium borate (LB). TAE has the
lowest buffering capacity but provides the best resolution for larger DNA. This means a lower
voltage and more time, but a better product. LB is relatively new and is ineffective in resolving
fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be
used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as
one base pair size difference could be resolved in 3 % agarose gel with an extremely low
conductivity medium (1 mM Lithium borate).
Analysis
After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a
light box, while using protective gear to limit exposure to ultraviolet radiation) to view the DNA
bands. The ethidium bromide fluoresces reddish-orange in the presence of DNA. The DNA band
can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel
can then be photographed usually with a digital or polaroid camera. Although the stained nucleic
acid fluoresces reddish-orange, images are usually shown in black and white (see figures).
Gel electrophoresis research often takes advantage of software-based image analysis tools, such
as ImageJ.
Typical method
Materials
Typically 10-30 μl/sample of the DNA fragments to separate are obtained, as well as a mixture
of DNA fragments (usually 10-20) of known size (after processing with DNA size markers either
from a commercial source or prepared manually).
 Buffer solution, usually TBE buffer or TAE 1.0x, pH 8.0
 Agarose
 An ultraviolet-fluorescent dye, ethidium bromide, (5.25 mg/ml in H2O). The stock
solution be careful handling this. Alternative dyes may be used, such as SYBR Green.
 Nitrile rubber gloves. Latex gloves do not protect well from ethidium bromide
 A color marker dye containing a low molecular weight dye such as "bromophenol blue"
(to enable tracking the progress of the electrophoresis) and glycerol (to make the DNA
solution denser so it will sink into the wells of the gel).
 A gel rack
 A "comb"
 Power Supply
 UV lamp or UV lightbox or other method to visualize DNA in the gel
Preparation
There are several methods for preparing gels. A common example is shown here. Other methods
might differ in the buffering system used, the sample size to be loaded, the total volume of the
gel (typically thickness is kept to a constant amount while length and breadth are varied as
needed). Most agarose gels used in modern biochemistry and molecular biology are prepared and
run horizontally.
 1. Make a 1% agarose solution in 100ml TAE, for typical DNA fragments (see figures). A
solution of up to 2-4% can be used if you analyze small DNA molecules, and for large
molecules, a solution as low as 0.7% can be used.
2. Carefully bring the solution just to the boil to dissolve the agarose, preferably in a
microwave oven.
3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or
swirl the solution while cooling.
Wear gloves from here on, ethidium bromide is a mutagen, for more information on safety see
ethidium bromide
1. Add 5 µl ethidium bromide (from a stock solution of 10 mg/ml) per 100 ml gel solution
for a final concentration of 0.5 µg/ml. Be very careful when handling the concentrated
stock. Some researchers prefer not to add ethidium bromide to the gel itself, instead
soaking the gel in an ethidium bromide solution after running, in this case the ethidium
bromide solution has final concentration of 0.5 µg/ml.
2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.
3. Insert the comb at one side of the gel, about 5–10 mm from the end of the gel.
4. When the gel has cooled down and become solid, carefully remove the comb. The holes
that remain in the gel are the wells or slots.
5. Put the gel, together with the rack, into a tank with TAE. Ethidium bromide at the same
concentration can be added to the buffer. The gel must be completely covered with TAE,
with the slots at the end electrode that will have the negative current.
Procedure
After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA
ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply
current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder
and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front
wave" approaches the end of the gel, the current is stopped. The DNA is stained with ethidium
bromide, and is then visible under ultraviolet light.
1. The agarose gel with three slots/wells (S).
2. Injection of DNA ladder (molecular weight markers) into the first slot.
3. DNA ladder injected. Injection of samples into the second and third slot.
4. A current is applied. The DNA moves toward the positive anode due to the negative
charges on its phosphate backbone.
5. Small DNA strands move fast, large DNA strands move slowly through the gel. The
DNA is not normally visible during this process, so the marker dye is added to the DNA
to avoid the DNA being run entirely off the gel. The marker dye has a low molecular
weight, and migrates faster than the DNA, so as long as the marker has not run past the
end of the gel, the DNA will still be in the gel.
6. Add the color marker dye to the DNA ladder.
Figure 1: Schematic drawing of the electrophoresis process, see text for description of steps