Gel Electrophoresis

GEL
ELECTROPHORESIS
BY: FAIZA RASHID
LECTURER
CITY UNIVERSITY OF SCIENCE AND INFORMATION
TECHNOLOGY, PESHAWAR, PAKISTAN
Gel electrophoresis
• Gel electrophoresis is a technique used to separate DNA fragments (or

other macromolecules, such as RNA and proteins) based on their size and
charge.
• Electrophoresis involves running a current through a gel containing the
molecules of interest (Charged molecules).
• Based on their size and charge, the molecules will travel through the gel in
different directions or at different speeds, allowing them to be separated
from one another.
• The movement of charged molecules is called migration. Molecules migrate
towards the opposite charge. A molecule with a negative charge will
therefore be pulled towards the positive end (opposites attract!).
Principle of gel electrophoresis
• The gel electrophoresis technique exploits the difference in size
and charge of different molecules in a sample. The DNA or protein
sample to be separated is loaded on to a porous gel placed in an
ionic buffer medium. On application of electric charge, each
molecule having different size and charge will move through the
gel at different speeds.
• The porous gel used in this technique acts as a molecular sieve
that separates bigger molecules from the smaller ones. Smaller
molecules move faster across the gel while the bulkier ones are
left behind. As a result the molecules are separated by size.
GEL ELECTROPHORESIS EQUIPMENT
Gel and buffer solution used in gel electrophoresis
• Gel electrophoresis involves a gel: a slab of Jelly-like material.

• The gel used in gel electrophoresis is usually made of a material polyacrylamide
or agarose.
• Agarose is a gelatinous substance extracted from seaweed or
• This porous gel could be used to separate macromolecules of many different
sizes.
• The gel is submerged in a salt buffer solution in an electrophoresis chamber. Tris-
borate-EDTA (TBE) is commonly used as the buffer. Its main function is to control
the pH of the system.
• Polyacrylamide gels are made by chemical polymerization of a mixture of
acrylamide and bisacrylamide (a cross-linker) in the presence of a catalyst and
an initiator of the polymerization reaction.
Gels used in gel electrophoresis
• Agarose is appropriate for separating DNA fragments ranging in size from a few
hundred base pairs to about 20 kb. Gels for DNA separation are often made out of
a polysaccharide called agarose, which comes as dry, powdered flakes. At the
molecular level, the gel is a matrix of agarose molecules that are held together by
hydrogen bonds and form tiny pores.
• Polyacrylamide is preferred for proteins and smaller DNA fragments.
• At one end, the gel has pocket-like indentations called wells, which are where the
DNA samples will be placed.
 The mobility of DNA is constant under defined electrophoretic conditions. These
conditions are characterized by the electrical parameters (current and voltage)
and factors such as buffer composition, agarose concentration, and temperature.
Gel box/ electrophoresis tank
• Before the DNA samples are added, the gel must be placed in a gel
box.
• One end of the box is hooked to a positive electrode, while the
other end is hooked to a negative electrode.
• The main body of the box, where the gel is placed, is filled with a
salt-containing buffer solution that can conduct current. The buffer
fills the gel box to a level where it just barely covers the gel.
• The end of the gel with the wells is positioned towards the negative
electrode. The end without wells (towards which the DNA fragments
will migrate) is positioned towards the positive electrode.
Gel box/ electrophoresis tank
How is gel electrophoresis carried out?
Preparing the gel
• Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used
to make the gel depends on the size of the DNA fragments you are working with.
• The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA
are separated on higher concentrations of agarose whilst larger molecules require a lower concentration
of agarose.
• To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high
temperature until all of the agarose powder has melted.
• The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make wells for
the sample to be pipetted into.
• Once the gel has cooled and solidified (it will now be opaque rather than clear) the comb is removed.
• The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank
until the surface of the gel is covered.
• The buffer conducts the electric current. The type of buffer used depends on the approximate size of the
DNA fragments in the sample.
Polyacrylamide gel for Polyacrylamide Gel electrophoresis PAGE
• Polyacrylamide is commonly known as PAGE is widely

used for separation of proteins and low molecular
weight DNA fragments. Polyacrylamide is the choice of
gel matrix in PAGE process.
• The gel is prepared between the two sandwiched glass
plates. Initially, solution for separation gel is poured in
between the glass plates, over which stalking gel is
layered. Comb is inserted immediately to cast wells.
Preparing the DNA for electrophoresis
• A dye is added to the sample of DNA prior to electrophoresis to increase the viscosity
of the sample which will prevent it from floating out of the wells and so that the
migration of the sample through the gel can be seen.
• A DNA marker (also known as a size standard or a DNA ladder) is loaded into the
first well of the gel. The fragments in the marker are of a known length so can be
used to help approximate the size of the fragments in the samples.
• The prepared DNA samples are then pipetted into the remaining wells of the gel.
• When this is done the lid is placed on the electrophoresis tank making sure that the
orientation of the gel and positive and negative electrodes is correct.
• In case of PAGE the polyacrylamide gel is removed and placed in the buffer filled
separating tank. Samples are mixed with tracking dye bromophenol blue and
sucrose. The mixed sample is then loaded (100 µl) in the wells along with markers.
Separating the fragments
• Once loading is complete, an electrical current of 50–150 V is

applied. Shorter lengths of DNA move faster than longer
lengths so move further in the time the current is run.
• The distance the DNA has migrated in the gel can be judged
visually by monitoring the migration of the loading buffer dye.
• The electrical current is left on long enough to ensure that the
DNA fragments move far enough across the gel to separate
them, but not so long that they run off the end of the gel.
• Electrophoresis is halted when the tracking dye is at another
end of gel.
Electrophoretic mobility
• The speed at which each molecule travels through the gel is called its
electrophoretic mobility and is determined mainly by its net charge
and size.
• Strongly charged molecules move faster than weakly charged ones.
Smaller molecules run faster leaving behind the larger ones.
• Thus, strong charge and small size increases a molecule’s
electrophoretic mobility, while weak charge and large size decreases
the mobility of a molecule.
• When all molecules in a sample are of the same size, the separation
will solely be based on their charge.
Illustration of Gel electrophoresis equipment
 Illustration of DNA
electrophoresis equipment used
to separate DNA fragments by
size.
 A gel sits within a tank of buffer.
 The DNA samples are placed in
wells at one end of the gel and
an electrical current passed
across the gel.
 The negatively-charged DNA
moves towards the postive
electrode.
Visualising the results
• Once the DNA has migrated far enough across the gel, the electrical current is
switched off and the gel is removed from the electrophoresis tank.
• Once the fragments have been separated, we can examine the gel and see what sizes
of bands are found on it.
• To visualise the DNA, the gel is stained with a fluorescent dye that binds to the DNA,
and is placed on an ultraviolet transilluminator which will show up the stained DNA as
bright bands allowing us to see the DNA present at different locations along the length
of the gel.
• Nucleic acids are generally stained by adding ethydium bromide directly to the
agarose gel or immersing agarose gel in the dye. Generally the gel is immersed in one
µg/ml solution of ethidium bromide for up to 10 minutes. Ethydium bromide quickly
spreads and concentrates wherever DNA molecules are present.
Visualising the results
• If the gel has run correctly the banding pattern of the DNA marker/size standard will be
visible.
• It is then possible to judge the size of the DNA in sample by imagining a horizontal line
running across from the bands of the DNA marker. The size of the DNA in the sample is
estimated by matching them against the closest band in the marker.
• A well-defined “line” of DNA on a gel is called a band. Each band contains a large
number of DNA fragments of the same size that have all travelled as a group to
the same position.
• Ethydium bromide intercalate between the bases of DNA molecules. The DNA appears as
fluorescent orange bands after exposed to ultraviolet from transilluminator.
• Nucleic acids can also be stained by soaking the gel in 0.025% solution of methylene blue
for 15 to 30 minutes. Methylene blue binds with DNA and appears as bluish bands under
normal room illumination.
 Illustration
showing DNA
bands separated
on a gel. The
length of the DNA
fragments is
compared to a
marker containing
fragments of
known length.
Applications of gel electrophoresis
• Gel electrophoresis is widely used in the molecular biology and biochemistry labs in areas
such as forensic science, molecular biology, and medicine. Some key applications of the
technique are listed below:
• In the separation of DNA fragments for DNA fingerprinting to investigate crime scenes
• To analyze results of polymerase chain reaction
• To analyze genes associated with a particular illness
• In DNA profiling for taxonomy studies to distinguish different species
• In paternity testing using DNA fingerprinting
• In the study of structure and function of proteins
• In the analysis of antibiotic resistance
• In blotting techniques for analysis of macromolecules
• In the study of evolutionary relationships by analyzing genetic similarity among populations
or species

Gel Electrophoresis

Uploaded by

Copyright:

Available Formats

Gel Electrophoresis

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Gel Electrophoresis

Uploaded by

Copyright:

Available Formats

GEL

• Gel electrophoresis is a technique used to separate DNA fragments (or

• Gel electrophoresis involves a gel: a slab of Jelly-like material.

• Polyacrylamide is commonly known as PAGE is widely

• Once loading is complete, an electrical current of 50–150 V is

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.