BIOC 21632
Experiment 04
Agarose gel analysis of DNA
Date:30/09/2022
Experiment No: 04
Experiment Name: Agarose gel analysis of DNA
Objectives: To demonstrate the separation of DNA fragments according to their size and
conformation during agarose gel electrophoresis.
Introduction:
An electrophoresis is an analytical tool that allows biochemists to examine the differential
movement of charged molecules in an electric field. A. Tiselius, a Swede who invented the
technique in the 1930s, performed experiments in free solutions that were severely limited by the
effects of diffusion and convection currents. Modern electrophoretic techniques use a
polymerized gel-like matrix, which is more stable as a support medium. The sample to be
analyzed is applied to the medium as a spot or thin band; hence, the term zonal electrophoresis is
often used.
The migration of molecules is influenced by:
(1) the size, shape, charge, and chemical composition of the molecules to be separated.
(2) the rigid, mazelike matrix of the gel support.
(3) the applied electric field.
Electrophoresis, which is a relatively rapid, inexpensive, and convenient technique, can analyze
and purify many different types of biomolecules but is especially effective with proteins and
nucleic acids.
THEORY OF ELECTROPHORESIS
Introduction The movement of a charged molecule in a medium subjected to an electric field is
represented by Equation
Were,
E = the electric field in volts/cm
q = the net charge on the molecule.
f = frictional coefficient, which depends on the mass and shape.
v = the velocity of the molecule.
where the electric field in volts/cm is the net charge on the molecule frictional coefficient, which
depends on the mass and shape of the molecule and the velocity of the molecule The charged
particle moves at a velocity that depends directly on the electric field (E) and charge (q), but
inversely on a counteracting force generated by the viscous drag (f). The applied voltage is
represented by E in Equation. held constant during electrophoresis, although some experiments
are run under conditions of constant current (where the voltage changes with resistance) or
constant power (the product of voltage and current). Under constant-voltage Conditions,
Equation 6.1 shows that the movement of a charged molecule depends only on the ratio For
molecules of similar conformation (for example, a collection of linear DNA fragments or
spherical proteins), f varies with size but not shape; therefore, the only remaining variables in
Equation 6.1 are the charge (q) and mass dependence off, meaning that under such conditions
molecules migrate in an electric field at a rate proportional to their charge-to-mass ratio.
Factors affecting the migration of DNA
Agarose concentration:
The mobility of DNA molecules is inversely proportional to gel concentration. Higher
percentage gels are sturdier and easier to handle but the mobility of molecules and staining will
take longer because of the tighter matrix of the gel. The most common agarose gel concentration
for separating dyes or DNA fragments is 0.8%. However, some experiments require agarose gels
with a higher percentage, such as 1% or 1.5%.
Size of DNA molecule
The sieving properties of the agarose gel influence the rate at which a molecule migrates. The
separation occurs because smaller molecules pass through the pores of the gel more easily than
larger ones. If the size of the two fragments is similar or identical, they will migrate together in
the gel.
DNA conformation
Different forms of DNA move through the gel at different rates; DNA molecules having a more
compact shape (e.g., plasmid DNA) moves faster through the gel compared with linear DNA
fragments of the same size. The migration rate of linear fragments of DNA is inversely
proportional to log 10 of their size in base pairs. This means that the smaller the linear fragment,
the faster it migrates through the gel.
Applied voltage
The mobility of DNA molecules is also affected by the applied voltage. Within a range, the
higher the applied voltage, the faster the sample migration.
Sample preparation and loading
Samples are prepared for electrophoresis by mixing them with loading dyes. Gel loading dye is
typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30%
glycerol). Loading dyes used in gel electrophoresis serve three major purposes:
1. add density to the sample, allowing it to sink into the gel.
2. provide color and simplify the loading process.
3. the dyes move at standard rates through the gel, allowing for the estimation of the
distance that DNA fragments have migrated.
4. These samples are delivered to the sample wells with a clean micropipette (a variable
automatic micropipette is the preferred one).
Ethidium bromide can be added to the gel during this step or the gel may also be stained after
electrophoresis in a running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by
destaining in a running buffer for an equal length of time
Applying electric current and separating biomolecules
A direct current (D.C.) power source is connected to the electrophoresis apparatus and an
electrical current is applied. Charged molecules in the sample enter the gel through the walls of
the wells. Molecules having a net negative charge migrate towards the positive electrode
(anode) while net positively charged molecules migrate towards the negative electrode
(cathode). The buffer serves as a conductor of electricity and controls the pH, which is important
to the charge and stability of biological molecules. Since DNA has a strong negative charge at
neutral pH, it migrates through the gel towards the positive electrode during electrophoresis.
The bluish-purple dye allows for visual tracking of sample migration during electrophoresis. The
gel is run until the dye has migrated to an appropriate distance.
Visualization
The agarose gel will have to be post-stained after electrophoresis. The most commonly used stain
for visualizing DNA is ethidium bromide (EtBr)*. Alternative stains for DNA in agarose gels
include SYBR Gold, SYBR green, crystal violet, and methyl blue. The sensitivities of methylene
blue and crystal violet are low compared with ethidium bromide. SYBR gold and SYBR green
are highly sensitive but more expensive than EtBr.
EtBr works by intercalating itself in the DNA molecule in a concentration-dependent manner.
When exposed to a short wave ultraviolet light source (transilluminator), electrons in the
aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light)
as the electrons return to the ground state. This allows for an estimation of the amount of DNA in
any particular DNA band based on its intensity. Ethidium bromide is a suspect mutagen and
carcinogen so must be handled cautiously. It is hazardous waste so must be disposed of
according to strict local and/or state guidelines. Stains containing methylene blue are considered
safer than ethidium bromide but should still be handled and disposed of with care.
The exact sizes of separated DNA fragments can be determined by plotting the log of the
molecular weight for the different bands of a DNA standard (DNA ladder) against the distance
traveled by each band. The DNA standard contains a mixture of DNA fragments of predetermined sizes that can be compared against unknown DNA samples.
Figure 01: Illustration of DNA electrophoresis equipment used to separate DNA
fragments by size. A gel sits within a tank of the buffer. The DNA samples are
placed in wells at one end of the gel and an electrical current is passed across the
gel. The negatively charged DNA moves toward the positive electrode.
Materials:
Figure : 02 Illustration showing DNA bands
separated on a gel. The length of the DNA
fragments is compared to a marker containing
fragments of known length.
Image credit: Genome Research Limited
Chemicals
Glassware
Apparatus others
Agarose
TBE buffer
Genomic DNA samples from
E -Coli
Loading dye
EtBr
ddH2O
Pipettes
Flasks
Microwave oven
Analytical balance
Mold
Electrophoresis chamber
Comb
Tapes
Eppendorf tubes
Centrifuge machine
Power supply
UV transilluminator
Methodology:
Preparation of the agarose gel
The gel former was sealed from the outside by tape. The comb was positioned about 1 mm
above the surface and agarose (0.8%) was poured into the gel tray. After the gel has hardened
completely (20 – 30 min), the comb was gently removed. The tape was removed, make sure the
gel tray was level to prevent the gel from sliding. The gel was transferred to the gel tray to the
electrophoretic apparatus and covered with TBE buffer. (50 mM Tris.20 mM NaOAc, 2mM
EDTA, pH 7.8). The DNA samples were mixed with a droplet of loading buffer (x 5, 50%
sucrose, 4mM urea, 50 mM EDTA, 0.1% bromophenol blue, 0.1% Orange G) in an Eppendorf
cap or on parafilm. The suitable amount of DNA strands was loaded in one well by using a
micropipette. Each sample was carefully loaded into a well. Each well was recorded. The
electrophoresis apparatus to the power supply and turn on the power supply. Agarose
concentration, Voltage used, distance migrated of the two dyes, and the time of electrophoresis
for each gel run was always recorded when the deep blue dye (Bromophenol blue) has migrated
to the bottom of the gel, and the gel proceeded to stain.
Staining the gel and Visualization DNA
The gel was stained for 20 min in ethidium bromide solution. After that gel was destined from
the water for 20 minutes. UV transilluminator box (302nm or 320nm) was wetted with water.
Then it was covered with plastic wrap. The plastic wrap prevented the ethidium bromide stain on
the UV box. The gel was carefully moved to the UV box. The plastic rulers were placed on each
side of the gel so that the distance each DNA band moved from the top of the well can be
estimated. After the UV protective glasses were placed, turn on the UV light, and the gel was
examined under UV light. The resulting photographs were used to determine the distance
migrated for each band. The plot is prepared of log10 (DNA fragment size ) against the distance
migrated. This plot is used to estimate the range of sizes of DNA that was migrated.
Results:
Agarose gel
Ladder
DNA fragments
RNA
.
Figure : Illustration showing DNA bands separated
Discussion:
Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic
acids. Agarose's high gel strength allows for the handling of low percentage gels for the
separation of large DNA fragments. Molecular sieving is determined by the size of pores
generated by the bundles of agarose7 in the gel matrix. In general, the higher the concentration of
agarose, the smaller the pore size. Traditional agarose gels are most effective at the separation of
DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one
will need to use pulse field gel electrophoresis6, which involves the application of alternating
current from two different directions. In this way, larger-sized DNA fragments are separated by
the speed at which they reorient themselves with the changes in the current direction. DNA
fragments smaller than 100 bp are more effectively separated using polyacrylamide gel
electrophoresis.
Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical-driven
chemical reaction. These thinner gels are of a higher concentration, run vertically, and have
better resolution. In modern DNA sequencing capillary electrophoresis is used, whereby
capillary tubes are filled with a gel matrix. The use of capillary tubes allows for the application
of high voltages, thereby enabling the separation of DNA fragments (and the determination of
DNA sequence) quickly.
EtBr is the most common reagent used to stain DNA in agarose gels. When exposed to UV light,
electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release
of energy (light) as the electrons return to the ground state. EtBr works by intercalating itself in
the DNA molecule in a concentration-dependent manner. This allows for an estimation of the
amount of DNA in any DNA band based on its intensity. Because of its positive charge, the use
of EtBr reduces the DNA migration rate by 15%. EtBr is a suspect mutagen and carcinogen;
therefore, one must exercise care when handling agarose gels containing it. In addition, EtBr is
considered hazardous waste and must be disposed of appropriately. Alternative stains for DNA
in agarose gels include SYBR Gold, SYBR green, Crystal Violet, and Methyl Blue. Of these,
Methyl Blue and Crystal Violet do not require exposure of the gel to UV light for visualization
of DNA bands, thereby reducing the probability of mutation if recovery of the DNA fragment
from the gel is desired. However, their sensitivities are lower than that of EtBr. SYBR gold and
SYBR green are both highly sensitive, UV-dependent dyes with lower toxicity than EtBr, but
they are considerably more expensive. Moreover, all the alternative dyes either cannot be or do
not work well when added directly to the gel, therefore the gel will have to be post-stained after
electrophoresis. Because of cost, ease of use, and sensitivity, EtBr remains the dye of choice for
many researchers. However, in certain situations, such as when hazardous waste disposal is
difficult or when young students are performing an experiment, a less toxic dye may be
preferred.
Loading dyes used in gel electrophoresis serve three major purposes. First, they add density to
the sample, allowing it to sink into the gel. Second, the dyes provide color and simplify the
loading process. Finally, the dyes move at standard rates through the gel, allowing for the
estimation of the distance that DNA fragments have migrated.
The exact sizes of separated DNA fragments can be determined by plotting the log of the
molecular weight for the different bands of a DNA standard against the distance traveled by each
band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can
be compared against unknown DNA samples. It is important to note that different forms of DNA
move through the gel at different rates. Supercoiled plasmid DNA, because of its compact
conformation, moves through the gel fastest, followed by a linear DNA fragment of the same
size, with the open circular form traveling the slowest.
Conclusion:
Agarose gel electrophoresis may be used to separate the DNA fragment which has different
sizes.
Reference:
Principles and Practice of Agarose Gel Electrophoresis Storage: Store the entire
experiment at room temperature EXPERIMENT OBJECTIVE. (n.d.).
https://people.wou.edu/~courtna/ch462/Gel%20Electrophoresis.pdf (Accessed
on 10/06/2022)
What is gel electrophoresis? (n.d.). @Yourgenome · Science Website.
https://www.yourgenome.org/facts/what-is-gel-electrophoresis/ (Accessed on
10/06/2022)
Discover the Microbes Within: The Wolbachia Project DNA Electrophoresis Lab-CIBT
Version AGAROSE GEL ELECTROPHORESIS LAB ACTIVITY AT A GLANCE. (n.d.).
https://cpb-use1.wpmucdn.com/blogs.cornell.edu/dist/3/1009/files/2015/05/WolbachiaLab-4-CIBT.pdf (Accessed on 10/06/2022)
Questions.
1.Describe briefly the basis of separation of DNA fragments according to their size during
agarose 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. 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.
All DNA molecules have the same amount of charge per mass. Because of this, gel
electrophoresis of DNA fragments separates them based on size only. Using electrophoresis, we
can see how many different DNA fragments are present in a sample and how large they are
relative to one another. Electrophoresis enables you to distinguish DNA fragments of different
lengths. DNA is negatively charged, therefore, when an electric current is applied to the gel,
DNA will migrate toward the positively charged electrode
2. What is the purpose of the gel loading buffer?
Gel loading buffer is used as a tracking dye during electrophoresis. The dye has a slightly
negative charge and will migrate in the same direction as DNA, allowing the user to
monitor the progress of molecules moving through the gel.
What is the purpose of the EtBr buffer?
Ethidium Bromide (EtBr) is sometimes added to the running buffer during the separation of
DNA fragments by agarose gel electrophoresis. It is used because upon binding the
molecule to the DNA and illumination with a UV light source, the DNA banding pattern
can be visualized.
What is the purpose of the buffer?
TAE Buffer (50X) is a solution used in Agarose Gel Electrophoresis (AGE) typically for the
separation of nucleic acids (i.e. DNA and RNA) and as a running buffer for preparative work.
What is the purpose of the molecular weight marker in agarose gel electrophoresis?
Molecular weight markers, or ladders, are a set of standards that are used for determining the
approximate size of a protein or a nucleic acid fragment run on an electrophoresis gel.
These standards contain a pre-determined fragment (or protein) sizes and concentrations.
What is the purpose of the gel comb marker in agarose gel electrophoresis?
The purpose of the comb in gel electrophoresis is to create wells to hold the samples. Before gel
electrophoresis, scientists need to cast the gel. While the gel is still liquid, a comb is set at the top
of the gel. This creates wells, or holes, that will hold the samples during electrophoresis.
What is the purpose of the Power supply marker in agarose gel electrophoresis?
Power supplies for electrophoresis are used to create the electrical current to power DNA/RNA
separations, PAGE electrophoresis, and transferred to the membrane. The voltage determines the
scope of function with 300V strong enough for running gels while 250V is needed to perform
transfers.
3. In Eykaryotes it is a chromosomal DNA larger than the plasmid DNA. In plasmid, it is
extrachromosomal DNA that is comparatively smaller. In human DNA it is essential for the
growth, survival, and reproduction of the cell. In the plasmid, it is not very essential for the
functioning of the cell. Human genomic DNA has a lower rate of replication while plasmid DNA
has a higher rate of replication. Genomic DNA is not used as a vector in recombinant DNA
technology while the plasmid is a popular cloning vector in recombinant DNA technology.
4.How do you extend agarose gel electrophoresis experiment to perform to prepare a
southen blot?
1.
2.
3.
4.
5.
6.
Digest the DNA with an appropriate restriction enzyme.
Run the digest on an agarose gel.
Denature the DNA (usually while it is still on the gel). ...
Transfer the denatured DNA to the membrane. ...
Probe the membrane with labeled ssDNA. ...
Visualize your radioactively labeled target sequence.
How do you extend the agarose gel electrophoresis experiment to isolate a DNA fragment
of known size from a mixture of DNA fragments?
A DNA marker with fragments of known lengths is usually run through the gel at the same time
as the samples. By comparing the bands of the DNA samples with those from the DNA marker,
you can work out the approximate length of the DNA fragments in the samples.
How do you extend the agarose gel electrophoresis experiment to construct a restriction
map of DNA Fragments?
One common method for constructing a restriction map involves digesting the unknown DNA
sample in three ways. Here, two portions of the DNA sample are individually digested with
different restriction enzymes, and a third portion of the DNA sample is double-digested with
both restriction enzymes at the same time.
5. Polyacrylamide gel electrophoresis provides very high resolution of DNA molecules 10–3,000
bp long. Under the appropriate conditions, DNA molecules differing in size by only a single base
pair can be resolved
6. What happens when the positive and negative electrodes are connected in the wrong way in
gel electrophoresis