Agarose Gel Electrophoresis

 

1. Preparation of Agarose Gel

DNA Agarose gels can be used to separate and visualize DNA of various sizes. Before casting an Agarose gel, consult Table-1 to determine the appropriate percent Agarose gel to use, based on the size of DNA to be separated.

 

Procedure

1. Determine the amount of Agarose (grams) required to make the desired Agarose gel concentration and volume. Example: For a 1% Agarose gel, add 1 gram of Agarose to 100 ml of 1x electrophoresis buffer.

 

      Table-1 Gel Concentration Required for DNA Separation

 

2. Add the Agarose to a suitable container (e.g., 250 ml Erlenmeyer flask, Wheaton bottle, etc.). Add the appropriate amount of 1x electrophoresis buffer and swirl to suspend the Agarose powder in the buffer.

Note: A mark can be put on the flask at the same level as the liquid. If evaporation occurs, water can be added to bring the liquid back to the original starting level.

 

3. Place the gel solution into the microwave oven and boil and swirl the solution until all of the small translucent Agarose particles are dissolved. Stopping the microwave oven and swirling the flask every 30 seconds helps dissolve the Agarose faster.

Caution: Always wear protective gloves, goggles, and a lab coat while preparing and casting Agarose gels. The vessels containing hot Agarose can cause severe burns if allowed to contact skin. Additionally, molten Agarose can boil over when swirled.

 

4. Cool the molten Agarose to 60 °C before pouring an Agarose gel slab.

Note: 0.5 µg/ml ethtidium bromide may be added to the molten Agarose. Alternative methods are described under Nucleic Acid Staining and Visualization.

 

 

2. Casting Agarose Gel

There are several ways to cast Agarose submarine gels using the Sub-Cell GT systems (BIO-RAD, Inc.). Gels may be cast with or without a UV-transparent plastic (UVTP) tray directly on the stage of the Sub-Cell GT bases using the gel casting gates. Gels may also be cast on the removable UVTP trays with the aid of the gel caster or with standard laboratory tape.

 

Removable tray (UVTP) gel casting using a Gel Caster or Mini-Gel Caster

1. Level the Gel Caster or Mini-Gel Caster using the leveling feet in the gel caster and the leveling bubble provided.

2. Disengage and slide the movable wall to the open end of the Gel Caster or Mini-Gel Caster by turning and lifting the cam peg upward.

Note: If casting more than one gel with the Gel Caster, add the removable gel casting wall to the gel caster. The removable wall will allow casting of two 15 x 10 cm trays, four 7 x10 cm trays or one 15 x 10 cm and one 15 x15 cm trays.

3. Place the open edge of the UVTP tray against the fixed wall of the Gel Caster or Mini-Gel Caster.

4. Slide the movable wall against the edge of the UVTP tray (Figure 1).

5. To seal the open tray ends, engage the cam peg by turning and pressing downward simultaneously.

6. When the cam peg has dropped into the appropriate slot, turn the peg in either direction until resistance is felt. This action seals the edges of the tray for casting.

7. Place the comb(s) into the appropriate slot(s) of the tray.

Figure 1. Sealing the UVTP tray for gel casting.

 

5. When the agarose solution has cooled to 50-60 °C, pour the molten Agarose on the tray.

Warning: Hot agarose (>60 °C) may cause the tray to warp or craze and will decrease the lifetime of the tray. Warping may also result in sample wells of uneven depth.

 

3. Setup and Run Electrophoresis

After the agarose gel has solidified, sample loading and electrophoresis can begin. Agarose gels can be run in many different types of electrophoresis buffers. Nucleic acid agarose gel electrophoresis is usually conducted with either Tris-Acetate-EDTA (TAE) buffer or Tris-Borate-EDTA (TBE) buffer. While TAE buffer provides faster electrophoretic migration of linear DNA and better resolution of supercoiled DNA, TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis runs.

1. Prepare samples for gel loading. Loading volume is dependent upon the type of comb used (i.e., well thickness and length) and thickness of the gel.

 

2. When loading volume is determined, add standard nucleic acid sample loading dye to a final 1x concentration to make samples dense for underlaying into sample wells

 

3. Load the samples into the wells using standard pipets.

Note: Sample wells are often difficult to see. Well visualization can be enhanced by placing black paper or tape under the base or trays where comb placement or well formation is common.

 

4. Place the lid on the DNA cell carefully. Do not disturb the samples. The Sub-Cell GT system lids attach to the base in only one orientation. To attach the lid correctly, match the red and black banana jacks on the lid with the red and black banana plugs of the base.

 

5. Power requirements vary depending on gel thickness, length and concentration, and type of electrophoresis buffer used. Use 20V to 110 V for most purpose. Refer to 2 for DNA size migration with sample loading dyes for the different Sub-Cell GT systems.

 

Table 2 DNA size migration with sample loading dyes

 

 

4. Gel Staining

There are different methods of staining nucleic acids for visualization using ethidium bromide.  A fast and sensitive method which is extensively used (although due to safety concerns, it is going out of favor) is ethidium bromide (EtBr) staining.  Three common applications include

  1. Adding 0.5 µg/ml directly to Agarose gel
  2. Adding 0.4 µg/ml directly into the sample and loading on EtBr-free gel
  3. Staining the gel after completion of electrophoresis

Caution: Ethidium bromide is a suspected carcinogen and should be handled with extreme care. Always wear gloves, eye glasses, and a laboratory coat. Dispose of used EtBr solutions and gels appropriately (Review EtBr Material Safety Data Sheet [MSDS] for proper disposal methods).

 

Ethidium Bromide Staining Procedure after Electrophoresis

1. Place the gel into the appropriate volume of 0.5 µg/ml ethidium bromide (EtBr) stain for 15–30 minutes. Use enough staining solution to cover the entire gel.

2. Destain the gel for 10–30 minutes in dH2O using the same volume used for staining.

Note: Ethidium Bromide can be removed from the DNA with extended destaining. This will cause lower sensitivity of detection. However, insufficient destaining will create higher background fluorescence.

3. Rinse the gel briefly with dH2O to remove any residual staining solution.

4. Place the gel on a UV transilluminator for nucleic acid visualization and analysis.

DNA/Ethidium Bromide complexes may be illuminated with UV light of 254, 302, or 366 nm. Sensitivity decreases with illumination at higher wavelengths. However, nicking of DNA will increase below 302 nm.

Note: Nucleic acids in the gel can be visualized through the UVTP trays. If a UVTP tray is not used, place household plastic wrap between the UV transilluminator and the gel to avoid contaminating the transilluminator with nucleic acids or EtBr.

 

5. Nucleic Acid Electrophoresis Buffers

DNA agarose gel electrophoresis is usually conducted with either Tris-Acetate-EDTA (TAE) or Tris-Boric Acid-EDTA (TBE). While TAE provides faster electrophoretic migration of linear DNA and better resolution of supercoiled DNA, TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis runs.

 

1x Tris-Acetate-EDTA (TAE)—40 mM tris (pH 7.6), 20 mM acetic acid, and 1 mM EDTA.

50x Stock (1 liter)—dissolve in 600 ml distilled water:

242 g tris base (FW = 121)

57.1 ml glacial acetic acid

100 ml 0.5 M EDTA (pH 8.0).

Fill to a final volume of 1 liter with distilled water.

 

1x Tris-Boric Acid-EDTA (TBE)—89 mM tris (pH 7.6), 89 mM boric acid, 2 mM EDTA

10x Stock (1 liter)—dissolve in 600 ml distilled water:

108 g tris base (FW = 121)

55 g boric acid (FW = 61.8)

40 ml 0.5 M EDTA (pH 8.0)

Fill to a final volume of 1 liter with distilled water.

 

DNA and RNA Sample Loading Dye

A convenient 10x sample buffer stock consists of 50% glycerol, 0.25% bromophenol blue, and 0.25% xylene cyanole FF in 1x TAE buffer. Only 1–10 ml of the 10x loading dye should be prepared.