DNA Electrophoresis on Agarose Gels

Making agarose gel stock (100 mL):

1. Weigh agarose powder on a balance. The amount of agarose needed depends on the concentration of the gel that will be used. The higher the concentration, the resolving ability is also higher.
2. For agarose gel 1% = 1 gram of agarose powder

1.5% = 1.5 grams of agarose powder

2% = 2 grams of agarose powder

3. Mix agarose powder with 1X TAE buffer in a heat-proof bottle. Dissolve it by heating in microwave oven on medium high for several minutes (use no lid or loosen lid while heating).
4. Occasionally during heating, shake the bottle to help the agarose dissolve.
5. Make sure the agarose dissolve completely (the mixture will be absolutely clear and show no traces of undissolved powder). Be careful not to overheat as the agarose mixture will overflow.
6. The agarose mixture is ready to be used directly or stored.

Electrophoresis preparation:

1. Prepare the comb and plate for pouring melted agarose. Use an appropriate comb depending on the number of samples that will be run.
2. Wait until the liquid agarose mixture cools down to around 60 ^oC (cool enough to not scorch your hand but not too cool that it partly solidifies). DO NOT POUR BOILING AGAROSE MIXTURE ON PLATE!
3. Pour agarose mixture on plate; the volume will depend on the volume of samples to be put in wells. For a reference, 30 mL of agarose mixture poured on plate with a standard 8-well comb will hold a maximum of 20µL of sample in each well.
4. Leave the agarose gel to set (around 10—15 minutes).
5. When the gel is set, take out the comb and put the plate in electrophoresis tank.
6. Pour enough 1X TAE buffer to cover the gel and fill the tank (approximately 250 mL buffer).

Loading samples and running electrophoresis:

1. Take out the samples, loading dye (2X), and DNA marker/ladder from fridge/freezer. Thaw completely.

2. Mix samples with loading dye on a parafilm sheet. As a guide, 2 µL of loading dye is enough to hold down 4—5 µL of sample. For larger amounts of samples, increase the amount of loading dye accordingly. Pipette up and down using a micropipette to mix the sample and loading dye completely.

3. Carefully pipette the sample/dye mixture into each well. After all samples are loaded into wells, load 4 µL of DNA ladder into one well. Note: the DNA ladder being used here is ready-to-use (already mixed with loading dye). If not, mix it with appropriate amount of loading dye first before pipetting it into the well.

4. Assemble the lid on tank and connect it to a power source. Make sure the side of gel that has wells is placed on the side of tank that is connected to the cathode (negative) of power source, as DNA is negatively charged and will travel to positively charged side (anode).

5. Set the current to maximum and set the voltage to 60-80 volts.

6. Run the electrophoresis for around 1—2 hours or until the loading dye reaches ¾ of gel (higher concentration of gel and lower voltage means longer running time). Do not let it run for too long or the DNA will be lost in the buffer.

Visualising DNA fragments:

1. When electrophoresis is finished, turn off power and open the tank. Take out the gel and put it on a container.
2. Pour EtBr (ethidium bromide) solution (30 µL in 500 mL of water) over the gel until it is completely soaked.
3. Soak the gel for 10—15 minutes. If the ethidium bromide solution is old or has been re-used many times, leave the gel soaked for longer.
4. Afterwards, pour back EtBr solution back in its bottle and wash the gel with tap water for 2—3 minutes.
5. Visualise DNA by viewing the gel under UV light.
6. If the DNA fragment is too faint, soak the gel again in EtBr solution. If the gel is too bright (soaked too long in EtBr), increase washing time to 5—6 minutes.
7. Photograph the gel with a manual polaroid camera or with GelDoc System.