Protocol: Running DNA Samples in Agarose Gel

OVERVIEW

  • Agarose gel electrophoresis is a very common method of analyzing DNA in most molecular biology laboratories.
  • DNA samples are mixed with DNA loading dye/buffer and loaded onto the wells of agarose gel. The DNA loading dye helps DNA samples to sink and settle to the bottom of the wells.
  • Since DNA is negatively charged due to its phosphate group, it moves towards the positive pole in the presence of an electric field.
  • Often a small amount of a suitable DNA staining dye such as ethidium bromide or GelRed is added to the agarose gel to visualize DNA. Alternatively, the gel can be briefly incubated in a solution of DNA staining dye to visualize DNA after electrophoresis.
  • The rate of the migration of DNA is primarily controlled by size and shape (conformation) of DNA, agarose concentration in the gel, and strength of the electric field (the voltage applied).
    • Small size DNA fragments move faster than larger fragments.
    • Generally, supercoiled DNA moves faster than linear DNA.
    • DNA fragments move faster in low percentage agarose gel. 
    • High voltage (high strength of electric field) causes faster movement of DNA fragments.
  • A higher percentage (2%) agarose gel is used to resolve small size DNA fragments whereas a lower percentage agarose gel is used to resolve large DNA fragments.
  • Generally, agarose gel electrophoresis is used to separate DNA fragments ranging from ≈ 75 bp to ≈ 30,000 bp (Sanderson et al., 2014).

REQUIREMENTS

Reagents and solutions
Agarose gel (see preparation of agarose gel)
6X DNA Loading Dye

Ethidium Bromide stock solution (10 mg/ml)
Electrophoresis buffer (TAE or TBE buffers)

Equipment and disposables
Micropipette and tips
Gloves
Electrophoresis Power supply
Electrophoresis apparatus
UV transilluminator or Gel doc

Note: Use the same electrophoresis buffer in which gel is prepared  (e.g., use TAE if the agarose gel is prepared in TAE).

PRECAUTIONS: Take appropriate safety measures and use personal protective equipment. Follow the guidelines of your institute and read the manuals/material safety data sheet of reagents and equipment used in this protocol. DNA staining dyes, especially Ethidium bromide, are carcinogens.

PROTOCOL

Step 1: Set the agarose gel in the electrophoresis tank in the right orientation.
Remove the tape from both sides of the casting tray (if you have used them for sealing the tray’s both open end).
Place the gel with a casting tray in the electrophoresis tank in the right orientation. Since DNA moves from negative to positive electrodes, the side where the wells are, should be towards the negative electrode.

Note
Different kinds of gel casting apparatus are available. In some cases, you need to seal the edges with the tape, whereas in others, you need to just place a casting tray into a cassette.

Precautions
1. If the casting tray is sealed with tape, remove the tape from both sides. Tape will not allow the passage of electric current through the gel.
2. Don’t remove the gel from the casting tray. This can cause damage to the wells.

Step 2: Fill the electrophoresis tank with an electrophoresis buffer.
The height of the buffer above the gel surface should not be more than 2 mm (Sanderson et al., 2014).

Tip
1. We recommend you to add ethidium bromide to a final concentration of 0.2 – 0.5 μg/ml in the electrophoresis buffer if the agarose gel contains ethidium bromide. For example, add 20 – 50 µl ethidium bromide (stock conc: 10 mg/ml)  in a 1000 ml electrophoresis buffer. As the electrophoresis progresses, positively charged ethidium bromide moves towards the negative electrode, resulting in the depletion of ethidium bromide from the rear end of the agarose gel. This results in the appearance of a dark zone in the agarose gel when you analyze the gel under the UV trans-illuminator.
2. Use the same electrophoresis buffer for running gel which was used to prepare agarose gel (e.g., Use TAE if the agarose gel was prepared in TAE).
3.
If you are using a TAE electrophoresis buffer, pour 1X TAE buffer in the tank. For TBE, use 0.5X TBE electrophoresis buffer (Sanderson et al., 2014).

Precautions
1. Agarose gel should be submerged in a buffer, but don’t fill electrophoresis buffer too much. Too much electrophoresis buffer over the agarose gel can cause slow run and distorted DNA bands.
2.
Ethidium bromide is carcinogenic. Use appropriate safety measures (wear latex gloves and lab coat) to avoid any harm.

Step 3: Carefully remove the comb from the gel without causing damage to wells.

Note
The comb can be removed even before placing the gel into the electrophoresis tank, but occasionally, this can cause collapsing of wells. It is much safer to remove the comb after submerging the gel in the electrophoresis buffer. The electrophoresis buffer moves inside the wells as the comb is removed, thus protecting the wells from collapsing.

Precaution:
Wells should not be damaged. Often wells are damaged at the bottom that may go unchecked, causing the leakage and loss of samples. If the samples are precious, check the well by loading a small amount of 1X loading dye onto the wells.

Step 4: Prepare DNA samples and load onto wells of agarose gel. 
Prepare DNA samples by mixing 5 volumes of DNA solution with 1 volume of 6X DNA loading dye (e.g., 5 μl DNA solution and 1 μl DNA loading dye). Use appropriate  micropipette to mix and load onto the wells of agarose gel carefully without spilling the sample into the adjacent wells.

Tips
1. Place the black paper beneath the electrophoresis apparatus to visualize wells in agarose gel.
2. If DNA samples need to be diluted, one can use water or more conveniently the electrophoresis buffer from the tank.
3.
It is very convenient and economical to mix the DNA solution with loading dye on the parafilm instead of using microcentrifuge tubes. To mix the sample with dye, one can place the required amount of dye on the parafilm. Now add a DNA solution and mix it by a few rounds (4 – 5 times) of pipetting.

Precautions
1. Do not destroy well while sample loading.
2. While pipetting DNA samples, avoid air bubbles. Air-bubbles can cause spillage of DNA samples into adjacent wells.

Step 5: Connect the electrophoretic apparatus to the power supply and start the gel run.
Place the lid on the gel box, and connect electrodes with a power supply using electric wires, supplied with the electrophoresis apparatus. Set the current maximum and voltage 70 – 100 volt. Turn the power supply on.

Notes
1. The distance between electrodes in the electrophoretic apparatus determines the maximum voltage. Gel prepared using standard agarose and common electrophoresis buffer (TBE or TAE) can be run at 5 V/cm. Since most commonly used electrophoresis devices have a distance 14 – 20 cm between electrodes, 70 – 100 V is the default voltage.
2. You can increase the voltage upto 10 V/cm to obtain a good resolution of small size DNA fragments by restricting their diffusion.

Tip
By looking at air-bubbles from the electrodes (negative electrode), one can ensure that the electric supply is functional.

Precautions
1. Always place the lid of the electrophoretic apparatus to avoid electric shock.
2. Make sure that connections [to positive (red in color) and negative poles (blue in color)] to the power supply are proper. If it is reversed, the sample will run in the opposite direction and will come out from the gel. The direction of the run can be monitored by observing the movement of the loading dye. (it will run in the same direction as the DNA).
3.
Remember that high voltage can cause uneven heating of gel and electrophoresis buffer that can result in slanting and broadening of the bands. In addition, faster movement of the samples in center lanes of the gel is also observed, which causes smiling of the bands (Sanderson et al., 2014). Excessing heating can also melt the gel.

Step 6: Run the agarose until Bromophenol blue approaches the end of the gel or 3/4 of the gel. Turn off the power supply and disconnect the wires.

Tip
Flow of current can be checked by looking at the bubbles forming on each electrode.

Note
Depending on the DNA size and resolution of DNA fragments, one has to decide the gel run time.

Step 7: Once the gel run is over, turn off the power supply and disconnect the wires. Take out the gel from the electrophoresis chamber and analyze it under UV transilluminator or Gel documentation system.

Note
Some casting trays allow direct visualization of gel as they are permeable to UV light. If the casting tray is impermeable to UV light, take out the gel and analyze under UV light.

Tip
Immediately analyze the gel for DNA just after you disconnect the electric supply. Storing gel in the electrophoresis buffer can cause loss of resolution due to diffusion of DNA bands.

Precautions:
1. Use hand gloves and follow safety rules as the gel contains ethidium bromide.
2. Use a tray to take out the gel. Some casting trays allow direct visualization of gel as they are permeable to UV light but are costlier. If the casting tray is impermeable to UV light, take out the gel, and analyze it under UV light.
3. UV is harmful. Use UV Safety Goggles and shield to protect yourself from direct exposure to UV rays.

REFERENCES

Sanderson et al., 2014. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis. Anal Biochem. 454, 44-52. PMID-24637158; Full-Text Links: sciencedirect, PMC4021863 (download PDF)

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