ELECTROPHORESIS: PREPARING AND RUNNING AGAROSE GELS
this module you will separate DNA molecules according to their size, using
agarose gel that you have prepared yourself.
However, before starting, please read the background material that
Electrophoresis is a method that
allows separation of molecules in an electric field on the basis of
size/molecular weight and shape. A
molecule will migrate in an electric field if it has a net positive or negative
charge. A molecule with negative
charge (anion) will migrate and be attracted to the positive electrode (anode),
and a molecule with positive charge (cation) will migrate and be attracted to
the negative electrode (cathode).
Migration and separation of molecules are carried out using pre-cast
“gels” composed of a solid matrix of molecules that contains microscopic
pores. Molecules are separated
according to size and shape by migrating through the pores in the gel. Separation is based on the different mobility through the gel
of molecules with different size and/or shape.
The gel matrix retards movement of molecules by a seiving effect.
Small or compact molecules travel faster through the gel matrix than
large or asymmetric molecules, which encounter more frictional resistance in the
Gel electrophoresis is a widely used technique in cell and molecular
biology for separating biological macromolecules such as nucleic acids (DNA,
RNA) and proteins. The gel matrix
for separating proteins is commonly polyacrylamide, a water-soluble cross-linked
polymer. Therefore, separation of
proteins is usually done by polyacrylamide gel electrophoresis (PAGE). Agarose, a complex linear polysaccharide, is routinely used
as a matrix component for separating DNA and RNA.
Therefore, this gel system is known as agarose gel electrophoresis.
Separation of protein on polyacrylamide gels and nucleic acids on agarose
gels generates banding patterns which can be visualized using various detection
The size of nucleic acid and protein molecules is typically measured in
different units. For DNA and RNA,
the length is conveniently measured in “base pairs” (bps) or kilobase pairs
(kbps). For proteins the
measurement is usually molecular weight in Daltons (Da) or kiloDaltons (kDa).
The sizes of DNA, RNA, or protein molecules separated on gels can be
estimated by comparison with standard molecular weight markers of known size
that were run in parallel with the unknown samples during gel electrophoresis.
The relative mobility of molecules, i.e., how fast they travel through
the gel relative to each other, depends on gel concentration as well.
Therefore, gels of differing concentrations, such as 1% and 2% agarose or
12% and 15% polyacrylamide, are used for separating different sized molecules.
The particular percent agarose or polyacrylamide needed depends on the
size of the molecules that require separation.
Generally, the smaller the size, the higher the percentage of agarose or
polyacrylamide needed. At a given
gel concentration, the distance traveled by a molecule will be inversely
proportional to the log10 of the
molecular weight or number of base pairs. Thus,
the larger or longer the molecule, the more time it takes to migrate through gel
matrices, as noted earlier. DNA and RNA molecules carry an intrinsic negative charge
conferred by their phosphate backbones. Proteins,
however, by virtue of the different charges on each amino acid residue, will
have different charges along their surface.
Therefore, for proteins, the mobility is proportional to molecular weight
only if a detergent such as sodium dodecyl sulfate (SDS) is used to coat the
protein surface and make charges uniformly negative along their length.
As noted above, DNA is a polyanion, carrying an innate negative charge
conferred by its negatively charged phosphates along the DNA backbone. Thus, in agarose gel electrophoresis, DNAS will migrate
through agarose from the negative cathode towards the positive anode.
Different size pieces of DNA will separate according to both their size
and shape. Lower molecular weight
(lower length) DNAs will move faster through gel matrix pores than larger ones.
However, the shape of a DNA molecule also plays a role in its movement,
with the fastest moving form known as “supercoiled” DNA.
It has the highest mobility because of the compactness of the
superhelical shape. Linear and
circular DNA molecules will travel slower because they tend to interact to a
greater extent with the gel matrix. Thus,
linear, circular, and supercoiled forms of DNA having the same number of base
pairs will migrate at different rates through agarose gels.
The resolution of DNA molecules by their size/shape using agarose gel
electrophoresis is truly remarkable. Good
separation can be achieved with DNA molecules whose size differs by as little as
1%, and it is also possible to separate two DNA molecules that differ by only a
single superhelical twist. Furthermore, the technique can be used with DNA
molecules containing fewer than 10 bps to as many as 300,000 bps.
Gels of different agarose concentration must be used for different size
- 1.5 % agarose for DNA sizes up to 50,000 bps
- 0.4% agarose for very large DNA sizes
While polyacrylamide gel electrophoresis is typically performed in a
vertical apparatus, agarose gel electrophoresis is conducted in a horizontal
configuration to provide better support at low agarose concentrations.
This produces less distortion (collapse) of the gel and of the DNA bands
during electrophoresis. The submarine system, in which the gel is completely
submerged in buffer, is the easiest to operate.
During electrophoresis water is electrolyzed, generating protons (H+)
at the anode, and hydroxyl ions (OH-)
at the cathode. The cathode end of
the electrophoresis chamber then becomes basic, and the anode end becomes
acidic. Use of a buffer system is
therefore needed. The two most
popular buffers for agarose gels are Tris-Borate-EDTA (TBE) and
tris-Acetate-EDTA (TAE). Besides
their excellent buffering capacity, they also aid in DNA mobility through the
In this module, you will use the TBE buffer and a 1% agarose gel, running
a mixture of dye markers as the sample. These
markers are test standards having properties resembling nucleic acids.
The “ChromatrackTM” dye markers are pipetted into one of the wells formed in the
agarose gel, then allowed to electrophorese.
The unique dye marker mix contains 6 dye markers that will migrate at
different molecular weights, providing a range of colored bands visible in
ordinary room light.
The following reference chart indicates the approximate bps with which
the individual tracking dyes migrate at different agarose gel concentrations.
Migration Chart (in bps)
#1: AGAROSE GEL ELECTROPHORESIS
View the video presentation on Agarose gel Electrophoresis
#2: LOCATING MATERIALS
Locate and check that you
have all of the following materials to perform agarose gel electrophoresis:
ii) agarose powder
i) 10X TBE buffer stock solution
ii) stirrer/heating unit
iii) power supply
v) P20 pipetman and pipet tips
vi) electrophoresis apparatus
(gel tank and lid)
gel electrophoresis drawer:
i) gel casting tray with foam
ii) gel running tray (detachable
from casting tray)
iii) comb to form wells
HIGH VOLTAGE IS USED IN ELECTROPHORESIS
#3: CASTING AND RUNNING AN AGAROSE
Weigh out agarose needed to make 30ml of a 1% gel.
(Reminder: % in this case is weight per unit volume, i.e, grams/100 ml)
Place the weighed-out agarose in a 150ml beaker.
Make up 280ml of 1X TBE buffer from stock 10X TBE.
The stock bottle is in the refrigerator.
Store buffer in a plastic storage bottle.
Add 30ml of the 1X TBE to agarose in the beaker
Add a stirring bar and place on stirrer/heating unit.
Set stirring to no. 2 and heating to no. 3.
Stir gently to a slight boil, when agarose dissolves and the liquid is
Carefully remove flask with paper towel or hot glove around neck of
flask, and place on bench. Remove stirring bar with magnet remover.
Allow gel to cool slightly. (All plastic parts of the electrophoresis
unit are heat resistant to 65oC, but
liquids should be cooled to about 50oC
before pouring in order to prolong unit life).
Use a thermometer.
Make sure that the gel running tray is safely tucked into the gel casting
tray, with tray edges gently pressed against foam pads.
Pour agarose into gel running tray
Add the comb to make wells, and allow the gel to solidify
Check that the power switch for the electrophoresis unit is OFF.
When gel hardens, carefully remove comb to expose wells
Remove the gel running tray from the casting tray and place it in the
electrophoresis unit gel tank
Add the rest of your 1X TBE so that it just completely covers the gel.
Do not use excessive buffer, as it will short-circuit the system and
lower the mobility
Using P20 pipetman, pipet 2µl of “ChromatrackTM”
into a middle well. Insert the
pipet tip into the bottom of the well and expel liquid slowly. Take care not to poke or puncture the agarose.
Connect the current electrodes (attached to the electrophoresis unit lid) so
that the dye markers will migrate toward the positive anode, i.e., connect
negative electrode at the position of the wells, and the positive electrode at
the other end of the gel.
Turn ON power supply and set to 100V. Band
migration of dye markers can be observed as electrophoresis proceeds.
The gel should be run for 1 hour, until all the colored bands are well separated
from each other. To se the bands,
turn OFF power supply, remove lid, and place a piece of white paper under the
gel tank to see the colored bands clearly.
Referring to the migration chart, identify the corresponding colored bands and
their approximate number of base pairs.
Remove the gel running tray (with gel in it) from the gel tank
Using a ruler, measure the distance traveled from the well (in cm) by each
23. Using semi-log paper, plot the number of base pairs of each colored band versus the distance traveled. plot base pairs on the log scale, and cm migrated on the linear scale. Draw the best line through these points to generate a standard curve.