XVI. ELECTROPHORESIS: PREPARING AND
RUNNING AGAROSE GELS
INTRODUCTION
In 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 follows.
Gel electrophoresis
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 meshwork.
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 methods.
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.
Agarose Gel Electrophoresis
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 ranges:
0.8 - 1.5 % agarose for DNA sizes up to 50,000 bps
0.2 - 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 trisAcetate-EDTA (TAE). Besides their excellent buffering
capacity, they also aid in DNA mobility through the gel
matrix.
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.
ChromatrackTM Migration Chart (in bps)
Dye
0.75%
blue 9,000
yellow
red
1.0%
4,300
2,600
1,300
4.0%
300
1,800
100
900
50
blue
950
600
pink
600
300
orange
200
100
25
15
<10
EXERCISE #1: AGAROSE GEL ELECTROPHORESIS VIDEO
View the video presentation on Agarose gel
Electrophoresis
EXERCISE #2: LOCATING MATERIALS
Locate and check that you have all of the
following materials to perform agarose gel
electrophoresis:
On reagent shelf:
i) Chroma TrackTM dye marker
ii) agarose powder
In refrigerator:
i) 10X TBE buffer stock solution
ii) stirrer/heating unit
iii) power supply
iv) balance
v) P20 pipetman and pipet tips
vi) electrophoresis apparatus
(gel tank and lid)
In gel electrophoresis drawer:
i) gel casting tray with foam pads
ii) gel running tray (detachable from casting tray)
iii) comb to form wells
CAUTION: HIGH VOLTAGE IS USED IN ELECTROPHORESIS
EXERCISE #3: CASTING AND RUNNING AN AGAROSE GEL
Procedure:
1. 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)
2.
Place the weighed-out agarose in a 150ml beaker.
3. 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.
4.
Add 30ml of the 1X TBE to agarose in the beaker
5. Add a stirring bar and place on stirrer/heating
unit. Set stirring to no. 2 and heating to no. 3.
6. Stir gently to a slight boil, when agarose
dissolves and the liquid is clear.
7. Carefully remove flask with paper towel or hot
glove around neck of flask, and place on bench.
Remove stirring bar with magnet remover.
8. 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.
9. Make sure that the gel running tray is safely
tucked into the gel casting tray, with tray edges
gently pressed against foam pads.
10. Pour agarose into gel running tray
11. Add the comb to make wells, and allow the gel
to solidify
12. Check that the power switch for the
electrophoresis unit is OFF.
13. When gel hardens, carefully remove comb to
expose wells
14. Remove the gel running tray from the casting
tray and place it in the electrophoresis unit gel
tank
15. 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
16. Using P20 pipetman, pipet 2µl of
“ChromatrackTM” dye marker 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.
17. 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.
18. Turn ON power supply and set to 100V. Band
migration of dye markers can be observed as
electrophoresis proceeds.
19. 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.
20. Referring to the migration chart, identify the
corresponding colored bands and their approximate
number of base pairs.
21. Remove the gel running tray (with gel in it)
from the gel tank
22. Using a ruler, measure the distance traveled
from the well (in cm) by each colored band.
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.