13.3: Lab Technique - Agarose Gel Electrophoresis of DNA

Separating DNA by Electrophoresis

Electrophoresis (meaning to “carry with electricity”) separates molecules based on their size using an electric field. In DNA agarose gel electrophoresis, fragments of DNA move through a polymer gel made of agarose. Agarose is a polysaccharide (primarily galactose) of an approximate molecular weight of 120,000 Daltons or 120 kDa (kiloDaltons). When solidified, it forms a three-dimensional "molecular sieve" made of pores with diameters ranging from 50 nm to >200 nm depending on the concentration of agarose used. Smaller fragments of DNA will pass more easily (and quicker) through the pores of the agarose gel than the larger fragments. Therefore, the rate at which a DNA fragment passes through the gel is inversely proportional to its size.

Preparation of the Agarose Gel

Agarose gels are prepared and run in a buffer that provides optimal salt concentration and pH for DNA separation. Most agarose gels are prepared in a TAE buffer (Tris-Cl, acetic acid, EDTA; pH of 8.0) because it offers the best separation of a large range of DNA fragment sizes (1 to 10 kb). It is also better if the DNA fragments need to be isolated from the agarose. However, the TAE buffer is prone to overheating so gels prepared in this buffer are usually run for a short duration (approximately 30 to 45 minutes). Agarose gels for the separation of smaller DNA fragments (less than 1 kb) can be prepared and run in a TBE Buffer (Tris-Cl, boric acid, EDTA). This buffer has a better buffering capacity and is more resistant to overheating. Therefore TBE-agarose gels are better for longer runs.

The DNA gel is made by pouring the prepared agarose solution into a mold, called a gel tray, containing a “comb” (Figure \(\PageIndex{1}\)). As the agarose cools, it polymerizes inside the tray and around the comb. When the comb is pulled from the polymerized gel, multiple wells are created for the addition of the DNA samples that will run through the gel in parallel “lanes”. A wide variety of gel tray sizes can be used in agarose electrophoresis, ranging from small trays at 5 x 6 cm to larger trays at 12 x 14 cm. The combs can also vary in size and number, providing the user with flexibility in terms of the number of DNA samples that can be run on each gel. The size of the gel trays and the combs that fit within them will depend on the manufacturer.

Figure \(\PageIndex{1}\): The DNA gel. The prepared agarose solution is poured into a tray and a comb inserted to create wells in the gel. (The DNA Gel by Patricia Zuk, CC BY 4.0)

An agarose gel of concentration between 1 to 2% can separate a wide range of DNA fragment sizes (50 bp to 10,000 base pairs). Most gels are prepared at a concentration of 1%. The higher the concentration of agarose, the smaller the pores and the better separation of smaller fragments will occur. However, concentrations greater than 2% may be more turbid, making it more difficult to visualize the DNA bands. Concentrations lower than 1% may be fragile and difficult to physically handle. Most manufacturers will have a table outlining the concentration of agarose in a gel and the range of DNA fragments it can separate. Gels may also be poured using Low Melting Point (LMP) agarose. This type of agarose will dissolve within the buffer at approximately 65°C. LMP agarose is more suitable for the analysis of DNA fragments greater that 10,000 bp (i.e., 10 kb).

Visualizing DNA within the agarose gel can be done by either adding a DNA dye to the agarose solution, or by adding the dye to the DNA loading buffer (see the next section). The most common method is the addition of ethidium bromide (EtBr) to the dissolved agarose solution. EtBr works by intercalating between the bases of the DNA. When exposed to UV light, EtBr glows orange, allowing the visualization of the DNA fragments. EtBr is carcinogenic because it can structurally distort the DNA helix and stop replication and transcription. As such, gels prepared with EtBR must be disposed of using correct biohazard procedures.

Preparation of DNA Samples

DNA samples are prepared for electrophoresis by mixing with a DNA loading buffer. A typical DNA loading buffer contains:

• salts to optimize ionic strength and pH for the DNA sample
• SDS or EDTA to inactive any enzymatic reactions associated with the DNA sample and to inactivate contaminating nucleases
• glycerol to make the DNA “heavy” so that it will sink to the bottom of the well
• one or more indicator dyes

The indicator dyes are necessary to track the progress of electrophoretic separation because DNA is colorless and cannot be seen in the gel. The indicator dyes will move through the agarose gel at different rates because, like the DNA fragments, they have different sizes. A typical DNA loading buffer will have at least two indicator dyes. The most commonly used are xylene cyanol (green color) and bromophenol blue (blue color), although alternate indicator dyes can be found in commercially-sold loading buffers. In an agarose gel of 1% prepared with TAE buffer, bromophenol blue will mimic a DNA fragment of about 350 bp while xylene cyanol runs as if it is approximately 4,000 bp. The distance that these two dyes will run is dependent on the agarose concentration and the buffer being used (Table \(\PageIndex{1}\)). The further the distance between these two dye "fronts" in the gel, the longer the gel has been running and the better resolution you will have between DNA fragments. Good resolution in a DNA gel is important so that individual DNA fragments of similar sizes can be distinguished from one another.

Table \(\PageIndex{1}\): Indicator Dye Sizes versus Agarose Concentration

Agarose Concentration (%)	Bromophenol Blue size in TAE Buffer (base pairs)	Bromophenol Blue size in TBE Buffer (base pairs)	Xylene cyanol size in TAE Buffer (base pairs)	Xylene Cyanol size in TBE Buffer (base pairs)
0.5	1,150	750	16,700	13,000
1.0	370	220	4,160	3,030
1.5	190	110	1,840	1,300
2.0	120	65	1,040	710

Most DNA loading buffers are purchased or made at either a 6X or 10X concentration. This will necessitate dilution of the loading buffer in the DNA sample so that it is at a 1X working concentration.

Determining the size of the separated DNA fragments requires the running of a DNA ladder alongside the DNA samples. A DNA ladder contains multiple DNA fragments where the size is known. A wide variety of DNA ladders can be purchased, ranging from a small-range "100 bp ladder" where the DNA fragments increase in size from 100 bp to 1000 bp in 100 bp increments, to larger scale ladders where the fragments can range from 100 bp to 15,000 bp. DNA ladders are typically sold already mixed with a loading buffer and are "ready to use".

Figure \(\PageIndex{2}\): DNA ladders. DNA samples are run alongside DNA ladders made up of DNA fragments of known sizes. DNA ladders can vary from small range (100 bp DNA ladder) to larger scale ladders made up of bigger DNA fragments (1kb DNA ladder). (DNA ladders by Patricia Zuk, CC BY 4.0)

Electrophoresis of DNA

To run the agarose gel, the gel is placed into an electrophoresis "tank" and the tank is filled with the same buffer used to make the gel, so as to create the electric field needed for electrophoretic separation. The tank will have an anode at one end (black electrode) and a cathode at the other end (red electrode). The electric field will flow through the TAE buffer in the tank in a circular pattern from anode to cathode and then back (Figure \(\PageIndex{3}\)). Because DNA is negatively charged, the DNA fragments will be pushed away from the negative anode (due to repulsion) and attracted toward the positive cathode. The voltage at which the gel is run is important. Too low and insufficient resolution between DNA fragments will result. Too high and the DNA fragments will run in a distorted pattern. The size of the gel, the voltage of the gel, and the anticipated sizes of the DNA fragments will all determine how long the gel should be run (Table \(\PageIndex{2}\)). Higher concentration gels will need less time. Running a gel at a higher voltage should translate into a short duration. In contrast, better separation between gel fragments will require longer run times.

Table \(\PageIndex{2}\): Summary of Gel Size and Voltage

DNA size range	Voltage	Optimal Running Buffer
less than 1 kb	5 to 10 V per cm gel length	TBE
1 to 5 kb	4 to 10 V per cm gel length	TAE or TBE
greater than 5 kb	1 to 3 V per cm gel length	TAE

Figure \(\PageIndex{3}\): DNA electrophoresis. The polymerized agarose gel is placed into an electrophoresis "tank" so that the sample wells are closest to the anode (the "-ve" electrode). A buffer is added to the tank and the electrodes connected to a power supply set to a specific voltage. The resulting electric field will result in the movement of the DNA towards the cathode (the "+ve" electrode). (DNA Electrophoresis by Patricia Zuk, CC BY 4.0)

Visualizing DNA Bands

DNA fragments within an agarose gel can be visualized using nucleic acid stains, fluorescent dyes, and even radioactive markers. The type of dye will determine the protocol used. For example, nucleic acid stains such as methylene blue are used after the gel has been run, while fluorescent dyes and radioactive markers are used "in-gel". In-gel staining, in which the dye is incorporated either into the agarose gel or added to the DNA sample via the loading buffer, is more convenient and requires significantly less dye. However, these dyes can only be used once and could alter the mobility of the DNA in the gel due to the binding of the dye to the DNA. Post-gel staining does not affect sample mobility. However, this method adds time to the workflow as individual staining and destaining steps will need to be performed. Moreover, some stains may not be as sensitive as the in-gel dyes.

As described above, the most common method of visualizing DNA fragments is the addition of the fluorescent dye ethidium bromide (EtBr) to the agarose solution. Today, the non-toxic fluorescent dye SYBR is a popular alternative to EtBr. Both dyes are fluorescent upon UV excitation. The intensity of fluorescence is direct proportional to the amount of DNA bound by the dye. EtBr is more sensitive than SYBR and can provide better visualization of smaller DNA bands. Visualizing both EtBR- and SYBR-stained DNA fragments will require the use of a UV transilluminator. The UV bulbs of a transilluminator are positioned below the gel and can provide short, medium and long-range UV wavelengths for visualization. UV light is extremely dangerous and proper safety protocols must be used to prevent damage to lab personnel. Many UV transilluminators come as part of a DNA visualization/documentation system in which the DNA gel is placed into a closed chamber containing the transilluminator (Figure \(\PageIndex{4}\)). In this way, the user is protected from UV exposure. A detector within the chamber provides an image of the gel on a screen. Exposure times can be adjusted to optimize the image of the gel and the image can be saved and printed out.

Figure \(\PageIndex{4}\): DNA documentation can be done by placing the gel on a UV transilluminator (A) or using a DNA documentation system, comprised of a UV transilluminator in a closed chamber connected to a monitor (B). (Agarose gel with ladders by Joseph Elsbernd, CC BY-SA 2.0; Gel Doc with PC by Bonnvenn, CC BY-SA 3.0)

Agarose Gel Electrophoresis Protocol

Lab Protocol: Preparing the Gel

1. For a 1% agarose gel, weigh out 1.0 g of electrophoretic grade agarose and add to a clean 125 mL glass flask.
2. Add 100 mL of 1X TAE buffer (or 1X TBE) to the flask and gently swirl to mix.
3. Heat the mixture to near boiling (90 to 95°C) to dissolve the agarose within the buffer. Ensure all agarose has been dissolved within the buffer. Allow to cool slightly.
4. Prepare the DNA tray according to the manufacturer's instructions.
5. Insert the comb at the top of the DNA tray.
6. Pour enough dissolve agarose solution for gel of approximately 5 mm thickness into a plastic centrifuge tube and allow to cool slightly. A volume between 40 and 50 mL is typically sufficient. Gels that are too thin will break upon handling. Gels that are too thick will produce "fuzzy" DNA bands. If using ethidium bromide to visualize the DNA bands, add enough ethidium bromide to this dissolved agarose sample for a final concentration of 0.5 ug/mL. Use correct biohazard procedures when handling the EtBr stock solution and agarose solution containing EtBR.
7. Carefully pour the slightly cooled agarose solution into the DNA tray.
8. Let stand at room temperature until the gel has completely polymerized. Making sure that the tray is level and will not be disturbed as it polymerizes.

**NOTE: Hot agarose is dangerous. Vigorously mixing hot agarose will cause it to boil over and may cause significant burns. As such, place the hot agarose mixture on the bench top and mix using a glass stir rod or long weighing scoop.

Lab Protocol: Sample Preparation

1. Depending on the size of the comb used, pipette the appropriate amount of DNA sample into a clean 1.5 mL microfuge tube. Most wells will accommodate volumes between 10 uL and 40 uL.
2. Add the correct volume of 10X DNA loading buffer to dilute its concentration to 1X. For example, if the DNA sample is 20 uL, add 2.0 uL of loading buffer.
3. Vortex to mix and flick spin to collect the contents at the bottom of the tube.

Lab Protocol: Running the Gel

1. Once the gel has fully polymerized, carefully pull out the comb to create the wells. Prepare the gel tray to remove the ends of the tray and to allow for buffer movement.
2. Place the tray in the electrophoresis tank so that the wells are closest to the black electrode.
3. Slowly fill the tank with 1X TAE or 1X TBE buffer. Fill the tank so that the entire surface of the gel is covered with the buffer. A “layer” of buffer on top of the gel with a depth of 3 to 5 mm is sufficient. Less than 3 mm could result in evaporative loss of buffer and drying of the gel. More than 5 mm of buffer will impede the electric field and the mobility of the DNA fragments in the gel.
4. Slowly pipette the DNA samples in the appropriate wells, being careful to introduce air bubbles into the well. In order to assess the size of the DNA fragments, it is important to pipette a DNA ladder into one of the wells.
5. Once the gel has been loaded, place the lid on the tank and hook it up to the power supply. Ensure that the black electrode (i.e., the anode) is closest to the DNA wells.
6. Set the voltage on the power supply to the desired voltage (see Table \(\PageIndex{2}\)).
7. Allow the gel to run so that the fastest running indicator due (e.g bromophenol blue) has traveled approximately 60 to 70% of the length of the gel.
8. Stop the gel when desired, remove the gel, and visualize the DNA fragments.

Lab Protocol: Visualizing DNA Bands "In-gel"

1. Prepare the agarose solution as detailed above.
   1. For EtBr staining, add enough to the dissolved agarose to a final concentration of 0.5 ug/mL.
   2. For SYBR staining, add the SYBR dye concentrate to the DNA loading buffer to a final concentration of 1X.
2. Load the DNA samples and DNA ladder and run the gel.
3. Carefully remove the gel and visualize the bands using a DNA documentation system or a UV transilluminator. If using a transilluminator, use proper UV safety protocols, including using a UV-blocking face shield and wearing a fully buttoned lab coat, long pants, and closed toe shoes. Make sure that all skin is protected from exposure to UV light, including face, neck, hands and arms.

Lab Protocol: Visualizing DNA Bands "Post-gel"

1. Prepare the agarose gel without any stains or dyes.
2. Run the DNA as detailed above.
3. Carefully remove the gel and place it into a staining container.
4. Add the desired stain and stain according to the manufacturer's specifications.
5. Remove the stain.
6. De-stain (if required) according to the manufacturer.
7. Visualize the bands using a DNA documentation system or a UV transilluminator.

Safety Considerations

• Hot agarose can cause burns. Do not vigorously mix hot agarose. Stir gently to dissolve agarose powder.
• Ethidium bromide (EtBr) is a carcinogen. Discard all materials containing EtBr using proper institutional guidelines.
• UV light can damage DNA. For UV transilluminators, use a UV-blocking face shield, a fully buttoned lab coat, long pants, and closed toe shoes. Make sure that all skin is protected from exposure to UV light, including face, neck, hands and arms.

Agarose Reagents

10X TAE (Tris, Acetate, EDTA)

• dissolve the following in 800 mL distilled water:
  • 48.4 g Tris base
  • 11.4 mL Glacial acetic acid
  • 7.4 g of disodium-EDTA
• adjust water to 1 L with distilled water (pH of the buffer will be approximately 8.0)
• store at room temperature
• final concentration:
  • 400 mM Tris
  • 200 mM acetate
  • 20 mM EDTA

1X TAE

• add 100 mL of 10X TAE to 900 mL of distilled water
• store at room temperature
• final concentration:
  • 40 mM Tris
  • 20 mM acetate
  • 2 mM EDTA

10X TBE (Tris, Borate, EDTA)

• dissolve the following in 800 mL distilled water:
  • 108 g Tris base
  • 55 g Boric acid
  • 9.3 g of disodium-EDTA
• adjust water to 1 L with distilled water (pH of the buffer will be approximately 8.3)
• store at room temperature
• final concentration:
  • 890 mM Tris
  • 890 mM boric acid
  • 20 mM EDTA

1X TBE

• add 100 mL of 10X TBE to 900 mL of distilled water
• store at room temperature
• final concentration:
  • 89 mM Tris
  • 89 mM boric acid
  • 2 mM EDTA

Ethidium Bromide (10 mg/ml)

• dissolve 200 mg ethidium bromide powder in 20 mL distilled water
• do not expose to light
• wear gloves
• discard gloves and weigh boat in a biohazard container
• store at room temperature

0.5M EDTA

• dissolve 186.1 g of disodium EDTA (Na₂-EDTA) in 400 mL distilled water
• pH to 8.0 with 10 N NaOH
• adjust to 1 L with distilled water
• store at room temperature

10X DNA Loading Buffer

• combine the following:
  • 2.5 mL 80% glycerol (20% final)
  • 0.5 mL of 10% SDS (1% final)
  • 0.2 mL of 0.5M EDTA (10 mM final)
  • 500 uL of 1% bromophenol blue (0.05% final)
  • 500 uL of 1% xylene cyanol (0.05% final)
• add nuclease-free water to 10 mL
• store at room temperature
• final concentration:
  • 20% final glycerol
  • 1% SDS
  • 10 mM EDTA
  • 0.05% bromophenol blue
  • 0.05% xylene cyanol