Topic: Agarose gel electrophoresis for the separation of
DNA fragments.
Agarose gel electrophoresis:
Agarose gel electrophoresis is a method of gel electrophoresis used
in biochemistry, molecular biology, genetics, and clinical chemistry to
separate a mixed population of macromolecules such as DNA or proteins in
a matrix of agarose, one of the two main components of agar. The proteins
may be separated by charge and/or size (isoelectric focusing agarose
electrophoresis is essentially size independent and
the DNA and RNA fragments by length. Biomolecules are separated by
applying an electric field to move the charged molecules through an
agarose matrix, and the biomolecules are separated by size in the agarose
gel matrix.
Material required:
The agarose powder, to make the gel.
Buffer stocks to make the running buffer.
Loading dye to mix with DNA
DNA Ladders to compare DNA lengths
DNA stain for visualising DNA
principle
The separation medium is a gel made from agarose. Agarose
is isolated from the seaweed
genera Gelidium and Gracilaria and consists of repeated
agarobiose (L- and D-galactose) subunits. During gelation,
agarose polymers associate non-covalently and form a
network of bundles whose pore sizes determine a gel’s
molecular sieving properties. In general, the higher the
concentration of agarose, the smaller the pore size. To
separate DNA using agarose gel electrophoresis, the DNA is
loaded into pre-cast wells in the gel and a current is applied.
The phosphate backbone of the DNA (and RNA) molecule is
negatively charged, therefore when placed in an electric
field, DNA fragments will migrate to the positively
charged anode. Because DNA has a uniform mass/charge
ratio, DNA molecules are separated by size.
Procedure:
1. Preparation of Agarose gel matrix
The centerpiece of agarose gel electrophoresis is the horizontal
gel electrophoresis apparatus. The gel is made by dissolving
agarose powder in a boiling buffer solution The concentration of
agarose in a gel depends on the sizes of the DNA fragments to be
separated, with most gels ranging between 0.5%-2%. The solution
is then cooled to approximately 55°C and poured into a casting
tray which serves as a mold. A well-former template (often called
a comb) is placed across the end of the casting tray to form wells
when the gel solution solidifies.
2). 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
These samples are delivered to the sample wells with a clean
micropipette (variable automatic micropipette is the preferred
one).
3) 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
4)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. 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 pre-determined
sizes that can be compared against the unknown DNA samples.
DNA concentrations can be estimated
by:
A. Taking absorbance at 260 nm.
At 260 nm, an absorbance (A) of 1 unit corresponds to a
concentration of: 50 μg/ml for dsDNA
40 μg/ml for RNA
33 μg/ml for ssDNA
20-30 µg/ml for oligonucleotides
Although this method is quick and nondestructive and gives
information about the purity of the sample (e.g., presence of
protein or organic contaminants), reliable estimates are obtained
only with concentrations of at least 1 μg/ml. Additionally, this
method cannot distinguish between DNA and RNA.
B. Intensity of Ethidium Bromide Fluorescence:
The amount of DNA in a sample can be estimated from the
intensity of ethidium bromide fluorescence (fluorescence
emitted by ethidium bromide is proportional to the amount of
DNA). The DNA quantity in an “unknown” solution can be
estimated by comparing its level of fluorescence with the
intensity of known amounts of DNA of similar size. This method is
useful if a DNA sample is contaminated with other compounds
that absorb in the UV range or is too dilute to measure at 260 nm.
Refrences:
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