Agarose Gel Electrophoresis of DNA

DNA is driven through the agarose matrix by electric current. Smaller or more compact molecules pass through the matrix easier and migrate farther than large molecules. All DNA has the same charge per unit length and linear pieces migrate according to size. The range of sizes separated in a gel is controlled by the % of agarose in the gel.

Resolution Versus Matrix Concentration
Agarose % in 1x TBE	Useful for Range of Linear dsDNA Molecules (kb)
0.3	5 - 60
0.6	1 - 20
0.7	0.8 - 10
0.9	0.5 - 0.7
1.2	0.4 - 6
1.5	0.2 - 3
2.0	0.1 - 2
*Information from Molecular Biology LabFax, ed. T. A. Brown, Academic Press, 1991

The mobility is proportional to the voltage applied at low voltage but increasing voltage decreases the resolution of larger fragments of DNA. A general guideline for agarose gels in 1xTBE is 5V/cm maximum for resolving fragment lengths greater than 2 kb. The distance between the electrodes serves as the length in the calculation. Higher voltages increase the temperature of the gel causing increased band width and distortion of the lanes. The agarose can also melt, especially the low melting point agarose sometimes used when DNA is to be recovered from the gel. The mobility is also influenced by the choice of buffer systems. Besides the Tris Borate EDTA, pH 8.3 (TBE) buffer used in our experiments, a Tris Acetate EDTA buffer (TAE) is preferred by some. The TAE buffer shifts the range of resolution toward higher fragment lengths.
Ethidium
Bromide

The nucleic acids are visualized with ethidium bromide (EtBr). This fluorescent dye, which contains a tricyclic planar group, intercalates between stacked base pairs of nucleotides and, in this environment, fluoresces when excited with ultraviolet light; the fixed position of the planar group and its close proximity to the bases causes dye bound to DNA to display increased fluorescent yield compared to free dye.

UV radiation at 254 nm is absorbed by DNA and transmitted to the dye, whereas radiation at 302 and 366 nm is absorbed by the bound dye
energy is re-emitted at 590 nm in the red-orange region of the visible spectrum

NOTE: most commercial UV light sources emit at 302 nm, which yields slightly less fluorescence than at 254 nm but produces LESS nicking of DNA.

We will include EtBr in the gel only. In this case the dye extends the length of linear and relaxed circular DNA by about 15% (the molecules are more rigid which decreases their mobility). Supercoiled DNA is positively supercoiled by ethidium bromide. Thus, the mobility of supercoiled DNA with respect to linear and relaxed circular DNA varies with the concentration of ethidium bromide present during the run.

If a DNA sample is too dilute to measure at 260 nm or is contaminated with other compounds that absorb in the UV range, the amount of DNA present can be estimated from the intensity of ethidium bromide fluorescence. Since the amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide, the DNA quantity in an "unknown" solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA.

The molecular weight DNA markers used in our study are Quick-Load® 1 kb DNA ladder (New England Biolabs, Catalog #N0468S). The ladder (see figure) produces eleven DNA fragments ranging in size from 500 to 10,000 base pairs (see picture). This ladder can be used to quantitate the amount of DNA in a sample; the mass of DNA in each band in the ladder has been calibrated.
The size of linear fragments of DNA is determined by comparison to standards: the log10 (# base pairs) is plotted versus distance migrated or Rf value {Helling R.B., Goodman H.M., and Boyer H.W. 1974. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol.14: 1235-1244}.
The loading buffer (LB) combined with the DNA samples contains two tracking dyes, *bromophenol blue* and *xylene cyanol, for visually monitoring electrophoresis and glycerol* to make the sample dense enough sink to the bottom of the well; the stock solution is designed to be diluted about six fold in the sample. Bromophenol blue runs about the same size as a linear double-stranded DNA molecule of 300 base pairs in length in 1X TBE on a gel of 1% agarose. In low percentage gels of 0.4% agarose, the dye can emulate a 1000bp fragment. Remember not to run this dye off the bottom of the gel when you are trying to analyze small fragments. Xylene cyanol runs about the same as a linear double-stranded DNA molecule of 4kb in a 1% agarose gel.

Agarose Gel Electrophoresis: Pouring and Running Gels

CAUTION

Agarose can become superheated and violently boil over. Exercise caution when heating. Swirl flask occasionally during heating. Heat until close inspection reveals that the agarose is 100% dissolved. Undissolved agarose will appear as little flecks that look like Lilliputian contact lenses.

HEALTH HAZARD

Ethidium bromide (EtBr) is a powerful mutagen and a probable carcinogen; it's moderately toxic and should be handled with care. Wear gloves when handling contaminated equipment or solutions containing ethidium bromide. Confine the compound to the restricted area. After running the gels, use paper towels and plastic wrap to protect equipment and surfaces from being contaminated. Dispose of the gels in the Biohazard Waste Box. Do NOT put paper towels, plastic wrap, or gloves in this waste box--ONLY the gels.

1.	Prepare gel tray as diagrammed using an adjustable gel caster. Level the tray using a bubble level.
2.	Flasks of melted agarose (0.8 or 1%) in 1X TBE have been prepared and held at 50°C. Carefully pour melted agarose (40-60 ml) into a beaker. Remember the solution is hot!
3.	Let the instructor know when you are ready to add the EtBr to your gel. After the instructor adds EtBr to the gel, gently swirl the agarose. EtBr stock solution = 0.625 mg/ml; we use 1 drop (~25 μl) EtBr/60 ml agarose so final concentration of EtBr = 0.3 μg/ml
4.	*Immediately, pour the melted agarose into the level* casting tray.** Use a pipet tip to push bubbles towards the bottom of the gel.
5.	Let the tray cool until gel is translucent (takes at least 20 minutes). CLEAN UP ANY DRIPS ON THE BENCH AND RINSE THE BEAKER WITH RO-H2O!
6.	Prepare DNA samples. For agarose gels it is advisable to load the same volume into each well. Some samples may need to be diluted with water or TE to achieve this. Thaw DNA at room temperature and pulse spin samples Add 6X loading buffer III (6X LB) to each sample for final concentration of 1X NOTE: 6X LB* contains* 0.25% bromophenol blue 0.25% xylene cyanol FF 30% glycerol (in water) from Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual*, Second Edition (Cold Spring Harbor Labortory Press)
7.	Turn lever on the caster to break the seal. Carefully remove comb and place casting tray into the electrophoresis unit for running. Fill unit with 1X TBE buffer to ~ 1 mm above gel. Pour buffer carefully onto the center of the gel to prevent the gel from sliding off the tray.
8.	Carefully load ALL of each sample into different wells in the gel and record the order of the samples in your notebook (record the location of each sample, not just the ones that you loaded). Do not press the tip into the bottom of the well while loading--allow the sample to sink there. The instructor or TA will load 10 µl of Quick-Load® 1 kb DNA ladder.
9.	Position the lid and connect the electrodes so that the anode is at the bottom of the gel (“run to red”). PLEASE NOTE: Banana plug fittings are not to be turned or twisted. Only push on and pull off by grasping the plug not the cord (or the entire lid for the boxes) without turning.
10.	Turn on the power supply (switch is located on the right side). Select “voltage” and set the voltage to 130 V using the raise/lower arrow keys; after the voltage is set, press the RIGHT SELECT button until DISPLAY is lit to monitor the actual voltage (v) and current (ma) during the run. Press RUN. The 500 bp standard will run just behind the dark blue dye front, and smaller fragments that run ahead of the dye may not be visible in this type of analysis. CAUTION: Lethal voltages are present while the power supply is "ON." Do not touch the gel or buffer until the power is off and electrodes are disconnected.
11.	STOP the run after ~ 45 min. Once the voltage is “zero” turn off the power supply, disconnect the electrodes, and carefully remove the lid.
12.	WEAR GLOVES. Drain excess buffer into the electrophoresis unit and place casting tray with gel onto a paper towel and carefully carry to the photography area. DO NOT spread EtBr outside the designated area!! From this point forward, assume that your gloves are contaminated with EtBr--do not touch anything that is not supposed to be contaminated. Place gel onto a sheet of plastic wrap on the transilluminator. CAUTION: The gel is still laden with EtBr and should be handled only with gloved hands. Scrupulously avoid all skin contact with the gel. Do not remove the gel from the designated EtBr bench. A waste container is provided there.
13.	Photograph the gel and compare the observed bands to the standards. The instructor or TA will take pictures for you using a UVP BioDoc-It™ System (components are listed below).

Gel Documentation

ATTENTION: Avoid doing anything that would unintentionally contaminate the transilluminator or anything else with EtBr. For instance, do NOT lay gels directly on the transilluminator, but always on plastic wrap. Do NOT contaminate the equipment (knob, cover, table, etc.)--REMOVE your gloves FIRST.

Position the gel in the center of the UV transilluminator and smooth out any wrinkles in the plastic wrap
With the cover in place, turn on the transilluminator
Capture an image of the gel with your Smart phone or camera

Dispose of the gels in the Biohazard Waste Box.
Do NOT put paper towels, plastic wrap, or gloves in this waste box--ONLY the gels.

Estimation of DNA Amounts

Two methods are widely used to estimate DNA concentrations.

Absorbance at 260 nm:At 260 nm, an absorbance (A) of 1 unit corresponds to a concentration of

50 μg/ml for dsDNA
40 μg/ml for RNA
33 μg/ml for ssDNA
20-30 µg/ml for oligonucleotides

Although this method is quick and nondestructive and gives information about the purity of the sample (e.g., presence of protein or organic contaminants), reliable estimates are obtained only with concentrations of at least 1 μg/ml. Additionally, this method cannot distinguish between DNA and RNA.

Intensity of Ethidium Bromide Fluorescence: If a DNA sample is too dilute to measure at 260 nm or is contaminated with other compounds that absorb in the UV range, the amount of DNA can be estimated from the intensity of ethidium bromide fluorescence. Since the amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide, the DNA quantity in an "unknown" solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA of similar size.

We would like to thank New England Biolabs for their generous support of our laboratory program

Copyright and Intended Use
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Created by David R. Caprette (caprette@rice.edu), Rice University14 Jul 08
Author: Beth Beason Abmayr, Ph.D., Rice University
Updated 17 January 2015