- Agarose gel electrophoresis is a very common method of analyzing DNA in most molecular biology laboratories.
- DNA sample is mixed with DNA loading dye/buffer and placed in wells of agarose gel. Loading buffer helps DNA sample to settle at the bottom of the wells.
- Since DNA is negatively charged due to its phosphate group, it moves towards positive pole under the electric field.
- Often a small amount of ethidium bromide is added in the agarose gel to visualize DNA. Alternatively gel can be placed in a ethidium bromide containing solution to visualize DNA after electrophoresis.
- Movement of DNA is primarily controlled by size and form (conformation) of DNA, agarose concentration in the gel and strength of electric field.
- Higher percentage (2%) agarose gel is used to resolve small size DNA fragments whereas lower percentage agarose gel is used to resolve large DNA fragments.
- DNA fragments lower than 50 bp are separated by polyacrylamide gel electrophoresis. Larger DNA fragments (more than 30 kB) are separated by pulse field gel electrophoresis.
- Preparation of agarose gel for DNA analysis
- Plasmid isolation by alkaline lysis method (miniprep)
- Preparation of 6X DNA loading dye (Bromophenol blue, xylene cyanol FF and Ficoll)
- Preparation of 6X DNA loading dye (Bromophenol blue, xylene cyanol FF and Sucrose)
- Preparation of 6X DNA loading dye (Bromophenol blue, xylene cyanol FF and Sucrose) Alternative method
- Preparation of DNase-free Bovine pancreatic RNase A
- Tracking dyes
- DNA loading dye
- Comparison of TAE and TBE electrophoresis buffer
- Agarose gel
- Agarose gel (see preparation process)
- Ethidium bromide solution (10 mg/ml in water)
- Micropipette and tips
- Electrophoresis Power supply
- Electrophoresis apparatus
- UV transilluminator or Gel doc
Note: Use the same electrophoresis buffer which is used to prepare agarose gel (e.g., Use TAE if the agarose gel is prepared in TAE).
Step 1: Place the agarose gel with casting tray in the electrophoresis tank. Since DNA moves from negative to positive electrode, the side where the wells are, should be towards the negative electrode.
- 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 casting tray into a cassette.
- If casting tray is sealed with tape, remove the tape from both sides. Tape will not allow the passage of electric current through gel.
- Don’t remove the gel from the casting tray. This may cause damage to the wells.
Step 2: Fill the electrophoresis tank with electrophoresis buffer.
- We recommend you to add ethidium bromide to a final concentration of 0.2 – 0.5 μg/ml in the electrophoresis buffer if the the agarose gel contains ethidium bromide. For example, add 20 – 50 µl ethidium bromide (stock conc 10 mg/ml) in 1000 ml electrophoresis buffer.
- As the electrophoresis progress, positively charged ethidium bromide moves towards negative electrode, resulting in depletion of ethidium bromide from the rear end of the agarose gel. This results in the appearance of dark zone in the agarose gel when you analyze gel under uv trans-illuminator.
- 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)
- If you are using TAE electrophoresis buffer, pour 1X TAE buffer in the tank. For TBE, use 0.5X TBE electrophoresis buffer.
- Agarose gel should be submerged in buffer, but don’t fill electrophoresis buffer too much. Too much electrophoresis buffer over the agarose gel can cause slow run and distorted DNA band.
- 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 the wells.
- The comb can be removed even before placing the gel in the electrophoresis buffer, but occasionally, this can cause collapsing of wells. It is much safer to remove the comb after submerging the gel in electrophoresis buffer. Electrophoresis buffer moves inside the wells as the comb is removed, thus protecting the wells from collapsing.
- Wells should not be damaged. Often wells are damaged at the bottom that may go uncheck, causing the leakage and loss of samples. If the samples are precious, check the well by loading a small amount of 1X loading dye in the wells.
Step 4: Prepare the DNA sample by mixing 5 volume of DNA solution with 1 volume of 6X DNA loading dye (e.g., 5 μl DNA solution and 1 μl DNA loading dye). Mix it and load into the wells of agarose gel carefully without spilling the sample into the adjacent wells.
- If DNA sample need to be diluted, one can use water or more conveniently the electrophoresis buffer from the tank.
- 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 DNA solution and mix it by few rounds (4 – 5 times) of pipetting.
- Do not destroy well while sample loading.
- While pipetting DNA sample, avoid air-bubbles. Air-bubbles can cause spillage of DNA sample into adjacent wells.
Step 5: Connect the electrophoretic apparatus to power supply and start the gel run.
- Place the lid on the gel box, and connect electrodes with power supply using electric wires, supplied with the electrophoresis apparatus. Set the current maximum and voltage 70 – 100 volt. Turn the power supply on.
- The distance between electrodes in electrophoretic apparatus determines the maximum voltage. Voltage should not exceed 5 volts/cm. Since most commonly used electrophoresis devices have distance 14 – 20 cm between electrodes, 70 – 100 volt is default voltage.
- By looking air-bubbles from the electrodes (negative electrode), one can ensure that the electric supply is functional.
- Always place the lid of electrophoretic apparatus to avoid electric shock.
- Make sure that connections [to positive (red in colour) and negative poles (blue in colour)] to power pack is proper. If it is reverse, sample will run on 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).
Step 6: Run the agarose until the purple dye (represents Bromophenol blue) approaches the end of the gel or 3/4 of the gel. Turn off the power supply and disconnect the wires from the power pack.
- Depending on the DNA size and resolution of DNA fragments, one has to decide the gel run time.
Step 7: Once the gel run over, turn off the power supply and disconnect the wires. Take out the gel from the electrophoresis chamber and analyze under UV transilluminator or Gel documentation system.
- Immediately analyze the gel for DNA just after you disconnect electric supply. Storing gel in electrophoresis buffer can cause loss of resolution due to diffusion of DNA band.
- Use hand gloves and follow safety rules as the gel contains ethidium bromide.
- Use tray to take out gel. Some casting trays allow direct visualization of gel as they are permeable to UV light but are costlier. If casting tray is impermeable to UV light, take out the gel and analyze under UV light.
- UV is harmful. Use UV Safety Goggles and shield to protect yourself from direct exposure to UV rays.