RESOLUTION AND RECOVERY OF SECTION II

DNA FRAGMENTS

Agarose Gel Electrophoresis UNIT 10.4

Agarose gel electrophoresis is a simple and highly effective method for separating,
identifying, and purifying 0.5- to 25-kb DNA fragments (see Finney, 1988, for resolution
of larger fragments by pulsed-field electrophoresis). The protocol can be divided into
three stages: (1) a gel is prepared with an agarose concentration appropriate for the size
of DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells
and the gel is run at a voltage and for a time period that will achieve optimal separation;
and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and
electrophoresis buffer, visualized directly upon illumination with UV light.

RESOLUTION OF DNA FRAGMENTS ON STANDARD AGAROSE GELS BASIC

PROTOCOL
Materials
Electrophoresis buffer (TAE or TBE)
Ethidium bromide solution
Electrophoresis-grade agarose
10× loading buffer
DNA molecular weight markers (Fig. 10.4.1)
Horizontal gel electrophoresis apparatus
Gel-casting platform
Gel combs (slot formers)
DC power supply

Lambda Lambda pBR322

BstEII (kb) Hin dIII (kb) BstNI (kb)

8.45
23.13
7.24
6.37 9.42
5.69 6.56
4.82 4.36
4.32
3.68
2.32 2.32
1.93 2.03 1.86
1.37
1.26 1.06
.93
.70
.56
.38
.22
.12 .13 .12

agarose concentration 1% buffer TAE

applied voltage 1V/cm gel run 16 hr

Figure 10.4.1 Migration pattern and fragment sizes of common DNA molecular weight markers.
Molecular Biology

10.4.1
Copyright © 1991 by Current Protocols Supplement 2 CPI
 Prepare the gel
1. Prepare an adequate volume of electrophoresis buffer (TAE or TBE; see commentary)
to fill the electrophoresis tank and prepare the gel.
To facilitate visualization of DNA fragments during the run, ethidium bromide solution can
be added to the electrophoresis buffer to a final concentration of 0.5 ìg/ml. If buffer is
prepared for the electrophoresis tank and the gel separately, be sure to bring both to an
identical concentration of ethidium bromide.
CAUTION: Ethidium bromide is a mutagen and potential carcinogen. Gloves should be
worn and care should be taken when handling ethidium bromide solutions.
2. Add the desired amount of electrophoresis-grade agarose to a volume of electropho-
resis buffer sufficient for constructing the gel (see Table 10.4.1 and commentary).
Melt the agarose in a microwave oven or autoclave and swirl to ensure even mixing.
Melted agarose should be cooled to 55°C in a water bath before pouring onto the gel
platform. This prevents warping of the gel apparatus. Gels are typically poured to a
thickness of 0.5 to 1 cm. Remember that the volume of the sample wells will be determined
by both the thickness of the gel and the size of the gel comb.
3. Seal the gel-casting platform if it is open at the ends. Pour in the melted agarose and
insert the gel comb, making sure that no bubbles are trapped underneath the combs
and that all bubbles on the surface of the agarose are removed before the gel sets.
Most gel platforms are sealed by taping the open ends with adhesive tape. As an added
measure to prevent leakage, hot agarose can be applied with a Pasteur pipet to the joints
and edges of the gel platform and allowed to harden.

Load and run the gel

4. After the gel has hardened, remove the tape from the open ends of the gel platform
and withdraw the gel comb, taking care not to tear the sample wells.
Most gel platforms are designed so that 0.5 to 1 mm of agarose remains between the bottom
of the comb and the base of the gel platform. This is usually sufficient to ensure that the
sample wells are completely sealed and to prevent tearing of the agarose upon removal of
the comb. Low percentage gels and gels made from low gelling/melting temperature
agarose should be cooled at 4°C to gain extra rigidity and prevent tearing.
5. Place the gel-casting platform containing the set gel in the electrophoresis tank. Add
sufficient electrophoresis buffer to cover the gel to a depth of about 1 mm (or just
until the tops of the wells are submerged). Make sure no air pockets are trapped within
the wells.

Table 10.4.1 Appropriate Agarose Concentrations

for Separating DNA Fragments of Various Sizes

Agarose Effective range of resolution of

(%) linear DNA fragments (kb)
0.5 1 to 30
0.7 0.8 to 12
1.0 0.5 to 10
1.2 0.4 to 7
1.5 0.2 to 3

Agarose Gel
Electrophoresis

10.4.2

Supplement 2 Current Protocols in Immunology

 6. Prepare DNA samples in a volume that will not overflow the gel wells by addition of
the appropriate amount of 10× loading buffer. Load samples into the wells using a
pipettor or micropipet, taking care not to mix samples between wells.
Be sure to include appropriate DNA molecular weight markers (see Fig. 10.4.1).
7. Be sure that the leads are attached so that the DNA will migrate into the gel toward
the anode or positive lead. Set the voltage to the desired level, typically 1 to 10 V/cm
of gel, to begin electrophoresis. Monitor the progress of the separation by visualizing
the migration of dyes (bromphenol blue) in the loading buffer.
If ethidium bromide has been incorporated into the gel and buffer, the separation can also
be monitored by viewing the gel with a hand-held UV lamp, which induces fluorescence of
ethidium bound to the DNA fragments in the gel.
CAUTION: To prevent electrical shocks, the gel apparatus should always be covered and
kept away from heavily used work spaces.
8. Turn off the power supply when the dye from the loading buffer has migrated a
distance judged sufficient for separation of the DNA fragments. If ethidium bromide
has been incorporated into the gel, the DNA can be visualized by placing the gel on
a UV light source and can be photographed directly (see support protocol).
Gels run in the absence of ethidium bromide can be stained by covering the gel in a dilute
solution of ethidium bromide (0.5 ìg/ml in water) and gently agitating for 10 to 30 min. If
necessary, destain by shaking in water for an additional 30 min. This serves to remove
excess ethidium bromide which causes background fluorescence and makes visualization
of small quantities of DNA difficult.

MINIGELS AND MIDIGELS SUPPORT

PROTOCOL
Small gels—minigels and midigels—can generally be run faster than larger gels and are
often employed to expedite analytical applications. Because they use narrower wells and
thinner gels, minigels and midigels also require smaller amounts of DNA for visualization
of the separated fragments. Aside from a scaling down of buffer and gel volumes, the
protocol for running minigels or midigels is similar to that described above for larger gels.
Similarly, the parameters affecting the mobility of DNA fragments, discussed below, are
the same for both large and small gels.
A number of manufacturers offer smaller versions of their large electrophoresis models.
An important feature to consider when selecting a mini- or midigel apparatus is the
volume of buffer held by the gel tank. As smaller gels are typically run at high voltages
(>10 V/cm), electrophoresis buffers are quickly exhausted, and it is therefore advanta-
geous to choose a gel apparatus with a relatively large buffer reservoir. Minigel boxes can
also be easily constructed from a few simple materials (Maniatis et al., 1982). A small
(e.g., 15 cm long × 8 cm wide × 4 cm high) plastic box can be equipped with male
connectors and platinum wire electrodes at both ends to serve as a minimal gel tank.
Although trays for casting small gels are commercially available, gels can also be poured
onto glass lantern slides or other small supports without side walls. Such gels are held on
the support simply by surface tension. After pouring the gel (e.g., 10 ml for a 5 cm × 8
cm gel), the comb is placed directly onto the support and held up by metal paper-binding
clamps placed to the side.
Since there is no agarose at the bottom of such wells, extra care must be taken to prevent
separation of the gel from the support when removing the comb.

Molecular Biology

10.4.3
Current Protocols in Immunology Supplement 30
 SUPPORT PHOTOGRAPHY OF DNA IN AGAROSE GELS
PROTOCOL DNA can be photographed in agarose gels stained with ethidium bromide by illumination
with UV light (>2500 µW/cm2). A UV transilluminator is typically used for this purpose,
and commercial models are available designed specifically for DNA visualization and
photography (UVP Inc.).
CAUTION: UV light is damaging to eyes and exposed skin. Protective eyewear should
be worn at all times while using a UV light source.
A bellows-type camera equipped with a Polaroid film holder provides a convenient means
for gel photography (see sketch 10.4A). An orange filter (Kodak Wratten #23A) is
required to achieve a desirable film image of light transmitted by fluorescing DNA. A
clear UV blocking filter (Kodak Wratten #2B) is also helpful. Polaroid type 667 film
(ASA 3000) offers ideal sensitivity, allowing as little as several nanograms of DNA to be
detected on film after making adjustments of exposure time.

REAGENTS AND SOLUTIONS

Agarose gel
Gels typically contain ∼1% agarose in 1× TAE or TBE buffer (see commentary).
Electrophoresis-grade agarose powder is added to 1× gel buffer and melted by
boiling for several minutes. Be sure all agarose particles are completely melted. To
facilitate visualization of DNA fragments during the run, ethidium bromide can be
added to 0.5 µg/ml in the gel.
Ethidium bromide solution
1000× stock solution, 0.5 mg/ml:
50 mg ethidium bromide
100 ml H2O
Dilute stock 1:1000 for gels or stain solution
Protect stock solution from light
10× loading buffer
20% Ficoll 400
0.1 M Na2EDTA, pH 8.0
1.0% SDS
0.25% bromphenol blue
0.25% xylene cyanol (optional; runs ∼50% as fast as bromphenol blue and can in-
terfere with visualization of bands of moderate molecular weight, but useful
for monitoring very long runs)
TAE electrophoresis buffer
50× stock solution:
57.1 ml glacial acetic acid

242 g Tris base 

(40 mM Tris⋅acetate)
57.1 ml glacial acetic acid
37.2 g Na2EDTA⋅2H2O (2 mM)
H2O to 1 liter
pH ∼8.5
TBE electrophoresis buffer
10× stock solution, 1 liter:
108 g Tris base (890 mM)
55 g boric acid (890 mM)
Agarose Gel
Electrophoresis 40 ml 0.5 M EDTA, pH 8.0 (20 mM)

10.4.4
Supplement 30 Current Protocols in Immunology
COMMENTARY
Background Information should be considered before undertaking an
Virtually all scientific investigations involv- electrophoretic separation. Parameters affect-
ing nucleic acids use agarose gel electrophore- ing the mobility of the DNA (i.e., applied voltage
sis as a fundamental tool. Two papers with and agarose concentration) should be chosen for
detailed descriptions of this technique and its optimal resolution of the desired DNA frag-
practical use for DNA analysis are McDonell ment(s). The protocol should be designed such
et al. (1977) and Southern (1979). that the progress of the electrophoretic separation
Voltage applied at the ends of an agarose gel can be monitored and the results accurately inter-
generates an electric field with a strength de- preted. This includes using such tools as tracking
fined by the length of the gel and the potential dyes and molecular weight markers.
difference at the ends (V/cm). DNA molecules
exposed to this electric field migrate toward the Parameters affecting the migration of DNA
anode due to the negatively charged phosphates through agarose gels
along the DNA backbone. The migration ve- Agarose concentration. Molecules of linear,
locity is limited by the frictional force imposed duplex DNA (form III) travel through gel ma-
by the gel matrix. While charge and/or size can trices at a rate that is inversely proportional to
affect the rate at which macromolecules will the log of their molecular weight (Helling et al.,
pass through a gel, the charge to mass ratio is 1974). The molecular weight of a fragment of
the same for DNA molecules of different lengths. interest can therefore be determined by com-
Therefore, it is the size of the DNA that deter- paring its mobility to the mobility of DNA
mines the rate at which it passes through the gel, standards of known molecular weight. This is
thereby allowing an effective separation of DNA the most valuable feature of agarose gel elec-
fragment-length mixtures by electrophoresis. trophoresis, as it provides a reproducible and
accurate means of characterizing DNA frag-
Critical Parameters ments by size.
Although agarose gel electrophoresis is Agarose concentration plays an important
straightforward and easy to perform, several factors role in electrophoretic separations, as it deter- Molecular Biology

10.4.5
Current Protocols in Immunology Supplement 2
 mines the size range of DNA molecules that trations and low applied voltages (∼1 V/cm,
can be adequately resolved. Figure 10.4.2 0.5% agarose).
shows the effects of agarose concentration on Electrophoresis buffers. The two most
mobility of fragments of different molecular widely used electrophoresis buffers are
weights. For most analyses, concentrations of Tris/acetate (TAE) and Tris/borate (TBE).
0.5% to 1.0% agarose are used to separate While these buffers have slightly different ef-
0.5-to 30-kb fragments. However, low agarose fects on DNA mobility (Fig. 10.4.2C), the pre-
concentrations (0.3% to 0.5%) are used to sepa- dominant factor that should be considered in
rate large DNA fragments (20 to 60 kb), and choosing between the two is their relative buff-
high agarose concentrations (1% to 1.5%) can ering capacity. Tris/acetate is the most com-
resolve small DNA fragments (0.2 to 0.5 kb). monly used buffer despite the fact that it is more
See also Table 10.4.1. easily exhausted during extended or high-volt-
Applied voltage. In general, DNA fragments age electrophoresis. Tris/borate has a signifi-
travel through agarose at a rate that is propor- cantly greater buffering capacity, but should be
tional to the applied voltage. With increasing avoided for purification of DNA from gels (see
voltages, however, large DNA molecules mi- gel purification protocols).
grate at a rate proportionately faster than small DNA conformation. Closed circular (form
DNA molecules. Consequently, higher volt- I), nicked circular (form II), and linear duplex
ages are significantly less effective in resolving (form III) DNA of the same molecular weight
large DNA fragments, as shown in Figure migrate at different rates through agarose gels
10.4.2. For separating large DNA molecules, it (Thorne, 1967). In the absence of ethidium
is best to run gels at both low agarose concen- bromide, closed circular supercoiled DNAs

pBR322, Form I (apparent molecular length)

pBR322, Form II (apparent molecular length) Xylene Cyanol

pBR322, Form III Bromphenol Blue

applied voltage 1 V/cm 0.5 2.5 gel 1% agarose

50 1 5 V/cm
buffer TAE buffer TAE
gel run 16hr mobilities determined
relative to 0.5 kb marker
10
5
log10 Molecular Length of DNA (kb)

1
0.5 0.5%
0.75%
A 1.0% B
1.5%

gel 1% agarose gel 1% agarose

50
applied voltage 1 V/cm applied voltage 1 V/cm
gel run 16 hr buffer TAE
10 gel run 16 hr
5 ± EtBr, 0.5 µg/ml

1
0.5 + EtBr
TAE - EtBr
C TBE D
2 4 6 8 10 12 2 4 6 8 10 12
Mobility (cm)

Agarose Gel Figure 10.4.2 Effect of (A) agarose concentration, (B) applied voltage, (C) electrophoresis buffer,
Electrophoresis
and (D) ethidium bromide on migration of DNA through agarose gels.
10.4.6

Supplement 2 Current Protocols in Immunology

such as plasmids migrate faster than their linear monitoring the progress of an electrophoretic
counterpart DNAs (Fig. 10.4.2D). Supercoil- separation is by following the migration of
ing essentially winds the molecules up, giving tracking dyes that are incorporated into the
them a smaller hydrodynamic radius and allow- loading buffer. Two widely used dyes display-
ing them to pass more readily through the gel ing different electrophoretic mobilities are
matrix. Nicked or relaxed circular molecules bromphenol blue and xylene cyanol. Xylene
that have lost all of their superhelicity migrate cyanol typically migrates with DNA fragments
appreciably slower than either supercoils or around 5 kb (Fig. 10.4.2A) and bromphenol
linear molecules (Fig. 10.4.2D) (Johnson and blue usually comigrates with DNA molecules
Grossman, 1977). around 0.5 kb (Fig. 10.4.1A). Bromphenol blue
The intercalating dye, ethidium bromide, is therefore provides an index of the mobility of
commonly incorporated into the gel and run- the fastest fragments and is particularly valu-
ning buffer. The dye reduces the mobility of able in determining the length of the gel over
linear duplexes (Fig. 10.4.2D) and has a par- which the separation of DNA has occurred.
ticularly pronounced effect on the mobility of Xylene cyanol is useful for monitoring the
closed circular DNA. Ethidium bromide progress of longer runs. Both dyes can interfere
changes the superhelical density of closed cir- with the visualization of the fragments that
cular molecules by inducing positive super- comigrate with them.
coils. With increasing concentrations of Ethidium bromide. Ethidium bromide is
ethidium bromide negative supercoils are commonly used for direct visualization of DNA
gradually removed, causing a concomitant de- in gels. The dye intercalates between the
crease in the mobility of the DNA molecule. stacked bases of nucleic acids and fluoresces
This occurs until a critical free-dye concentra- red-orange (560 nm) when illuminated with
tion is reached where no more superhelical UV light (260 to 360 nm). This allows detection
turns remain (usually between 0.1 and 0.5 of very small quantities of DNA (<5 ng) (Sharp
µg/ml). As still more ethidium bromide is et al., 1973).
bound, positive superhelical turns are gener- Ethidium bromide is frequently added to the
ated which, like negative supercoils, cause an gel and running buffer prior to electrophoresis.
increase in the electrophoretic mobility of the While this has a slight effect on the mobility of
molecules (i.e., Fig. 10.4.2D). By running gels the DNA (Fig. 10.4.2D), it eliminates the need
at different concentrations of ethidium bro- to stain the gel upon completion of the separa-
mide, therefore, form I DNAs can easily be tion. An added advantage to running gels with
distinguished from other topoisomers. ethidium bromide is that the mobility of the
DNA can be monitored throughout the run until
Monitoring and interpreting separations of the desired separation is achieved.
DNA through agarose gels Molecular weight markers. Among the sam-
The DNA sample. In choosing the amount ples loaded onto a gel, at least one lane should
of DNA to be loaded, the width of the well plus contain a series of DNA fragments of known
the depth of the gel and the number and size of sizes so that a standard curve can be constructed
DNA fragments should be considered. Be- to allow the calculation of the sizes of unknown
tween 5 and 200 ng of a single DNA fragment DNA fragments. The most commonly used
can be loaded into a 0.5-cm-wide × 0.2-cm- molecular weight markers are restriction di-
deep sample well; 5 ng approaches the minimal gests of phage λ DNA or, for smaller fragments,
amount of an individual DNA fragment detect- the plasmid pBR322. Figure 10.4.1 shows the
able by ethidium bromide staining, and 200 ng migration pattern and fragment sizes for restric-
approximates the most that can be resolved tion digests of l DNA and pBR322 that are
before overloading occurs (the trailing and frequently used as molecular weight markers.
smearing characteristic of overloading become Aside from λ restriction fragments, many
more pronounced with DNA fragments above commercial preparations of molecular weight
10 kb). For samples of DNA containing several markers are also available. These products usu-
fragments, between 0.1 and 0.5 µg of DNA is ally cover a wide range of DNA sizes, and some
typically loaded per 0.5 cm sample well. Up to manufacturers also offer supercoiled (form I)
10 µg of DNA can be adequately resolved for markers for calculating plasmid sizes.
samples containing numerous fragments of dif-
ferent sizes (e.g., restriction digests of genomic Troubleshooting
DNA). Common problems encountered in agarose
Tracking dyes. The most common means of gel electrophoresis are described below, along Molecular Biology

10.4.7
Current Protocols in Immunology Supplement 2
 with several possible causes. including ethidium bromide in the gel and
Poor resolution of DNA fragments. The monitoring the progress of the run directly by
most frequent cause of poor DNA resolution is visualization with UV light.
improper choice of agarose concentration. Mini- and midigels are typically run at high
Low–percentage-agarose gels should be used voltages relative to gel size (>10 V/cm) and are
to resolve high-molecular-weight DNA frag- often completed in <1 hr. Two consequences of
ments and high-percentage gels for low-mo- high-voltage runs should be kept in mind: first,
lecular-weight DNAs (see Table 10.4.1). Fuzzy as mentioned above, buffers become quickly
bands, encountered particularly with small exhausted and therefore TBE should be used,
DNA fragments, result from diffusion of the or gel tanks with large buffer capacities. Sec-
DNA through the gel. This is especially true ond, high voltages provide poor resolution of
when gels are run for long periods of time at high-molecular-weight DNA fragments (see
low voltages. previous discussion). If large DNA fragments
Band smearing. Trailing and smearing of are to be separated, it is advisable to use larger
DNA bands is most frequently observed with gels and/or lower applied voltages.
high-molecular-weight DNA fragments. This
is often caused by overloading the DNA sample Literature Cited
or running gels at high voltages. DNA samples Finney, M. 1988. Pulsed-field gel electrophoresis.
loaded into torn sample wells will also cause In Current Protocols in Molecular Biology (F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore,
extensive smearing, as the DNA will tend to run
J.G. Seidman, J.A. Smith, K. Struhl, eds.) pp.
in the interface between the agarose and the gel 2.5.9-2.5.15. Greene Publishing and Wiley-In-
support. terscience, New York.
Melting of the gel. Melting of an agarose Helling, R.B., Goodman, H.M., and Boyer, H.W.
gel during an electrophoretic separation is a 1974. Analysis of EcoRI fragments of DNA from
sign that either the electrophoresis buffer has lambdoid bacteriophages and other viruses by
been omitted in the preparation of the gel or has agarose gel electrophoresis. J. Virol. 14:1235-
1244.
become exhausted during the course of the run.
For high-voltage electrophoresis over long time Johnson, P.H. and Grossman, L.I. 1977. Electropho-
resis of DNA in agarose gels. Optimizing sepa-
periods, TBE should be used instead of TAE as
rations of conformational isomers of double-and
it has a greater buffering capacity. Also, minigel single-stranded DNAs. Biochemistry 16:4217-
and midigel boxes, which typically have small 4224.
buffer reservoirs, tend to exhaust buffers more Maniatis, T., Fritsch, E.F., and Sambrook, J. 1989.
readily. Molecular Cloning: A Laboratory Manual (2nd
ed.). Cold Spring Harbor Laboratory, Cold
Anticipated Results Spring Harbor, N.Y.
DNA fragments of ∼0.5 to 25 kb are well Sharp, P.A., Sugden, B., and Sambrook, J. 1973.
resolved using the basic protocol. It is neces- Detection of two restriction endonuclease activi-
ties in Haemophilus parainfluenzae using ana-
sary to use pulsed-field gel electrophoresis for
lytical agarose. Biochemistry 12:3055-3063.
resolving molecules from ∼10 to >2000 kb
Thorne, H.V. 1967. Electrophoretic characterization
(Finney, 1988).
and fractionation of polyoma virus DNA. J. Mol.
Biol. 24:203.
Time Considerations
The parameter that has the greatest effect on Key References
the length of time required to complete an McDonell, M.W., Simon, M.N., and Studier, F.W.
electrophoretic separation is the applied volt- 1977. Analysis of restriction fragments of T7
age. Most large agarose gels are run overnight DNA and determination of molecular weights by
electrophoresis in neutral and alkaline gels. J.
(∼16 hr) at between 1 and 1.5 V/cm. While gels Mol. Biol. 110:119-146.
can be run much faster, particularly if the gel
Southern, E. 1979. Gel electrophoresis of restriction
is cooled, resolution of larger DNA frag-
fragments. Methods Enzymol. 68:152-176.
ments is lost due to the higher voltages. Since
the resolution required depends on the rela-
tive molecular weights of the fragments of
Contributed by Daniel Voytas
interest, the time required for adequate sepa-
Iowa State University
ration is best determined empirically. As de- Ames, Iowa
scribed above, this is most easily done by
Agarose Gel
Electrophoresis

10.4.8

Supplement 2 Current Protocols in Immunology