Principles of Gel

Electrophoresis
• A day without electrophoresis is very rare in
molecular biology labs, because this
technique is the standard method used for
analyzing, identifying and purifying
fragments of DNA. It is also used for
separating and analyzing RNAs and
oligonucleotides.
• Electrophoresis is a technique used to
separate and purify macromolecules -
especially proteins and nucleic acids - that
differ in size, charge or conformation.
• When charged molecules are placed in an
electric field, they migrate toward either the
positive (anode) or negative (cathode) pole
according to their charge.
• Proteins have either a net positive or net
negative charge, nucleic acids have a
consistent negative charge (phosphate
backbone) and migrate toward the anode.
• Proteins and nucleic acids are
electrophoresed within a matrix or "gel" in
the shape of a thin slab, with wells for
loading the sample.
• The gel is immersed within an
electrophoresis buffer that provides ions to
carry a current and some type of buffer to
maintain the pH at a relatively constant
value. ( Movie )
 The gel itself is composed of either agarose
or polyacrylamide, each of which have
attributes suitable to particular tasks:
• Agarose is a polysaccharide 0.5 to 2%w/v.
Simply mix agarose powder with buffer
solution, melt it by heating, and pour the gel
( physical process) . It is non-toxic. It is a
relatively smooth gel.
A large range but relatively low resolving power
of separation. Varying agarose concentrations
separate DNA fragments from about 100 to
50,000 bp.
Agarose Range of separation of linear DNA
(%) (in kilobases)
0.3 60 - 5
0.6 20 - 1
0.7 10 - 0.8
0.9 7 - 0.5
1.2 6 - 0.4
1.5 4 - 0.2
2.0 3 - 0.1
• Polyacrylamide is a cross-linked polymer of
acrylamide (typically between 3.5 and 20%
acrylamide) (chemical between acrylamide and
bis-acrylamide) .
Annoying to prepare where oxygen inhibits the
polymerization therefore poured between glass
plates (or cylinders). A hard gel and easy to
recover in pure state.
Acrylamide powder itself is a potent
neurotoxin.
A rather small range but very high resolving
power separation, separate molecules that
differ in size by as little as 2% of their molecular
weight .
Separating DNA fragments of less than about
100 bp. DNA fragments differing is length by
a single base pair are easily resolved. (Therfore
,for proteins, used extensively for separating
and characterizing).
 Percent Acrylamide Size Range
((19:1 bis-acrylamide (# of basepairs)

20 6-100

15 25-150

12 40-200

8 60-400

5 80-500

3.5 1000-2000
 The equipment and supplies necessary for
conducting agarose gel electrophoresis (or
PAGE)are relatively simple and include:
• An electrophoresis chamber and power supply
• Gel casting trays
• Sample combs to form sample wells in the gel
• Electrophoresis buffer, as Tris-acetate-EDTA
• Loading buffer and one or two tracking dyes
e.g, Bromophenol blue and xylene cyanol (for
monitoring)
• Ethidium bromide, a fluorescent dye used for
staining
• An ultraviolet lightbox
3. Preparation of the gel
4. Microwave oven, casting tray, ethidium
bromide (or soaking after electrophoresis),
sample combs, cooling
5. Assembling, loading, and power on.
6. Migration and visualization (ultraviolet
lightbox).
Figure 1
Optimizing factors in gel separation:

a. Agarose (or polyacrylamide)

concentration: Higher concentrations of
agarose (or polyacrylamide) facilite separation
of small DNAs
 Figure 2
using same gel tray, .same voltage, same running time
b. Voltage: As the voltage is increased larger
fragments migrate proportionally faster than
small fragments.

c. Electrophoresis buffer: Buffers not only

establish a pH, but provide ions to support
conductivity. DNA fragments will migrate at
somewhat different rates in two buffers. But
concentrated buffer could generate heat that
causes gel to melt.
The rate of migration is determined by

(1) the charge on the macromolecule, (2) the

strength of the electrical field, (3) the size of the
macromolecule, (4) the shape of the
macromolecule, (5) the state of hydration of the
macromolecule, (6) the viscosity of the solution,
and (7) the pore size of the gel.
where v = the rate (velocity) of migration, E is
the strength of the electrical field, z is the
charge on the molecule and f is the frictional
force on the molecule.

where η is the viscosity of the medium and r is

the stokes radius of the molecule.
The gel serves two purposes. It serves to diffuse
convective currents and to creates a molecular
sieve (enhancing the separation based on
molecular weight).

Note: since nucleic acids are negatively charged

urea –and not EDTA- is used to denature the DNA
or RNA within the gel.

Mobility -distance from the well- is inversely

proportional to the log10 of their molecular
weight or number of base pairs (straight
line).
Figure 3
DNA can be isolated from Agarose gel by
different methods e.g Electroelution : Cut out
the desired piece into dialysis tubing with a
small amount of fresh electrophoresis buffer.
Current will cause the DNA to migrate out of
the agarose. Then DNA is precipated out by
ethanol.

DNA is isolated from polyacrylamide gel by

cutting out the desired piece into elution buffer,
incubation then spinning down the gel. Then
DNA is precipated out by ethanol.
Southern blotting
• Double-stranded DNA on the gel can be
separated into single strands by denaturation in
alkaline solution.
• DNA is then (or at the same time) moved from
the gel onto membrane (nitrocellulose or nylon)
by pressuring the membrane and gel with a
stack of paper towels. DNA will stick and
permanently crosslink to the membrane by
special treatment.
• The membrane is then treated with a
hybridization probe which is an isolated DNA
(or RNA) molecule with a specific sequence
that pairs with the sequence we are looking for.
• The probe DNA has to be labelled so that it can
be detected, usually by incorporating
radioactivity or tagging the molecule with a
fluorescent or chromogenic dye.

• Excess probe is washed from the membrane,

and the pattern of hybridization is visualized on
x-ray film by autoradiography.
Figure 4 Blotting
(Figure 5 e-d steps)
DNA is extracted from the cells and purified. A
large piece of DNA is chopped into smaller
pieces using a restriction enzyme. The DNA is
loaded into a well #2of the gel matrix. Well #1
contains standards of known DNA fragments to
quantitate the size.Well #3 contains
unrestricted (intact) DNA .
Figure 6 A-B
Northern blotting
A technique used to study gene expression. Similar to
southern blotting with the key difference that RNA,
rather than DNA, is the substance being analyzed by
electrophoresis and detection with a hybridization
probe.
Formaldehyde is used to denature DNA on gel ,
because the sodium hydroxide treatment used in the
southern blot procedure would degrade the RNA.
The hybridization probe may be made from DNA or
RNA.
A variant of the procedure known as the Reverse
Northern Blotting : a collection of isolated DNA
fragments is affixed to the membrane and the probe is
RNA extracted from a tissue and radioactively labelled
(DNA microarrays).
Applications
DNA Fingerprinting. e.g.the identification of particular
DNA molecules by the band patterns they yield in gel
electrophoresis after being cut with various restriction
enzymes (Viral DNA, plasmid DNA, and particular
segments of chromosomal DNA can all be identified).
DNA Recombinant Technology. e.g. the isolation and
purification of individual fragments containing
interesting genes, which can be recovered from the
gel with full biological activity …
The Human Genome Project. e.g. to map the entire
human genome and to determine the genetic
difference and the evolutionary relationship among
species of plants and animals.