Agarose Gel Electrophoresis APPENDIX 4F

RESOLUTION OF LARGE DNA FRAGMENTS ON AGAROSE GELS BASIC

PROTOCOL
This protocol is used to separate and purify DNA fragments between 0.5 and 25 kb.
Materials
Electrophoresis buffer (TAE or TBE; APPENDIX 2E)
Ethidium bromide solution (APPENDIX 2E)
Agarose, electrophoresis-grade
10× loading buffer: 20% (w/v) Ficoll 400/0.1 M disodium EDTA, pH 8.0/1%
(w/v) SDS/0.25% (w/v) bromphenol blue
DNA molecular weight markers (Fig. A.4F.1)
Horizontal gel electrophoresis apparatus
Gel casting platform
Gel combs (slot formers)
DC power supply
1. Prepare the gel, using electrophoresis buffer and electrophoresis-grade agarose (see
Table A.4F.1) by melting in a microwave oven or autoclave, mixing, cooling to 55°C,
pouring into a sealed gel casting platform, and inserting the gel comb.
Ethidium bromide can be added to the gel and electrophoresis buffer at 0.5 µg/ml.
CAUTION: Ethidium bromide is a potential carcinogen. Wear gloves when handling.

Lambda Lambda pBR322

Bst EII (kb) Hin dIII (kb) Bst NI (kb)

8.45
23.13
7.24
9.42
6.37
6.56
5.69
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 A.4F.1 Migration pattern and fragment sizes for commonly used DNA molecular weight
markers.
Molecular
Biology
Techniques

Contributed by Daniel Voytas A.4F.1

Current Protocols in Protein Science (1998) A.4F.1-A.4F.3
Copyright © 1998 by John Wiley & Sons, Inc. Supplement 13
 Table A.4F.1 Appropriate Agarose Concentrations
for Separating DNA Fragments of Various Sizes

Agarose Effective range of resolution

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

2. After the gel has hardened, remove the seal from the gel casting platform and
withdraw the gel comb. Place into an electrophoresis tank containing sufficient
electrophoresis buffer to cover the gel ∼1 mm.
3. Prepare DNA samples with an appropriate amount of 10× loading buffer and load
samples into wells with a pipettor. Be sure to include appropriate DNA molecular
weight markers (Fig. A.4F.1).
4. Attach the leads so that the DNA migrates to the anode or positive lead and
electrophorese at 1 to 10 V/cm of gel.
5. Turn off the power supply when the bromphenol blue dye from the loading buffer has
migrated a distance judged sufficient for separation of the DNA fragments.
Bromphenol blue comigrates with ∼0.5-kb fragments.
6. Photograph a stained gel directly on a UV transilluminator or first stain with 0.5 µg/ml
ethidium bromide 10 to 30 min, destaining 30 min in water, if necessary.

SUPPORT MINIGELS AND MIDIGELS

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 for larger gels. Similarly, the
parameters affecting the mobility of DNA fragments are the same for both large and small
gels.
When selecting a mini- or midigel apparatus consider the volume of buffer held by the
gel tank. Smaller gels are typically run at high voltages (>10 V/cm) so electrophoresis
buffers are quickly exhausted; therefore, it is advantageous to choose a gel apparatus with
a relatively large buffer reservoir. Minigel boxes can also be easily constructed from a
few simple materials (Sambrook et al., 1989). 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
Purification and cm gel), the comb is placed directly onto the support and held up by metal paper-binding
Concentration of clamps placed to the side. There is no agarose at the bottom of such wells, so extra care
DNA from
Aqueous Solutions must be taken to prevent separation of the gel from the support when removing the comb.

A.4F.2
Supplement 13 Current Protocols in Protein Science
LITERATURE CITED
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Southern, E. 1979. Gel electrophoresis of restriction fragments. Methods Enzymol. 68:152-176.

Contributed by Daniel Voytas

Iowa State University
Ames, Iowa

Molecular
Biology
Techniques

A.4F.3
Current Protocols in Protein Science Supplement 13