Agarose Gel Electrophoresis

of DNA Fragments

Overview:
Gel Electrophoresis is a procedure used in molecular biology to separate
and identify molecules (such as DNA and RNA) by size. The separation of
these molecules is achieved by placing them in a gel made up of small
pores and setting an electric field across the gel.
The molecules will move based on their inherent electric charge
(i.e., negatively charged molecules move away from the negative pole) and
smaller molecules will move faster than larger molecules; thus, a size
separation is achieved within the pool of molecules running through the gel.
The gel works in a similar manner to a sieve separating particles by size;
the electrophoresis works to move the particles, using their inherent electric
charge, through the sieve.

Learning objectives:
- Visualize, identify and distinguish molecules that have been processed by
a previous method such as PCR, enzymatic digestion or an experimental
condition.

Personal Protective Equipment:

- Wear a long-sleeved lab coat, safety goggles, nitrile gloves, long pants,
and closed-toe shoes.
- Wear appropriate skin and eye protection when working with UV radiation
and ethidium bromide.

Tools and materials:

- Agarose powder.
- 1x TAE buffer.
- 400 ml graduated container.
- Microwave oven.
- Ethidium bromide dye.
- Pipettes.
- Gel tray.
- Casting gates.
- Electronic balance.
- Comb.
- Gel tank.
- PCR products (DNA samples).
- 6x Gel loading buffer.
- DNA pre stained ladder.
- Power supply.
- UV transilluminator.

Procedure:
Stage 1: Agarose preparation
1. Weight 2 gm of agarose powder.

2. Prepare 200 ml 1x TAE buffer in a 400 ml graduated container.

3. Add the 2 gm agarose powder to the 200 ml 1x TAE buffer and shake the
graduated container.
Note: We have worked on 1% agarose which matches with our DNA
expected size.

4. Heat the agarose and 1xTAE buffer mixture in microwave oven for 1:30
min.

Caution:
- Make sure the container holding the gel mixture is large enough to allow
the solution boil up without coming out of the container.
- A loose cover should be placed on the top to prevent the solution from
splashing out, the cover should be loose otherwise dangerous pressure
build up can occur within the container.

5. Pause the microwave oven at the last 00:30 sec and take the container
out and shake it then enter it the microwave oven again.

Caution:
- Make sure you wear safety gloves at this step because the solution can
become superheated and when you mix it the liquid can suddenly boil
up.

6. Put the agarose mixture again in the microwave oven and complete the
last 00:30 sec.

7. Get the agarose mixture from the microwave oven and make sure the
agarose is “completely dissolved”
i.e: the agarose mixture should be completely transparent.

8. Leave the agarose to cool to 60 °C to prevent the heat damage of the gel
tray.
9. Add 3 μl ethidium bromide dye to molten agarose gel using pipette.

Caution:
- When dealing with ethidium bromide you should wear gloves, eye and
skin protection because it is carcinogenic and you should get rid of the
pipette and the gloves once you add it to the molten gel to avoid
contamination of other parts of the lab.

Stage 2: Casting the gel

10. Take the gel tray and close opening using casting gates.

11. Insert the comb into the gel tray.

12. Take agarose gel container and slowly pour the gel solution into the gel
tray.

Caution:
- Using new pipette tip to move any air bubbles to the edge of the gel, it is
important to remove bubbles from around the comb since these can
affect the shapes of the wells.

13. Leave the gel 20 min to solidify.

14. Remove the casting gates from the ends of the gel tray.

Stage 3: Setting up the electrophoresis chamber

15. Put the gel tray in the gel tank and make sure the wells are in the side
of the –ve electrode (black electrode).

16. Add 1x TAE buffer solution to each end of the gel tank and keep adding
buffer until the surface of the buffer is 2 mm above the surface of the
wells in the gel. (this will facilitate the gel loading).

17. Slowly pull the comb up from the gel.

18. Place a dark back ground under the gel tank in order to see the well
loading obviously.

19. Label the samples.

20. Mix 10 μl from DNA sample with 2 μl from 6x gel loading buffer.
Note: The loading buffer will help us to track the migrating samples.
21. Mix the gel loading buffer with the sample by stirring using pipette tip
in order to get uniform color solution.

Caution:
- Avoid using the wells at the edges of the gel because samples loaded at
the edges tend to run less consistently than samples loaded near the
center.

Stage 4: Loading the gel

22. Slowly Load the samples to the gel wells by taking 10 μl of each sample
using pipette and add to the well.

Caution:
- Be careful not to blow air in the well once the sample is being expelled.
- Before loading the sample to the well make sure there is no air bubbles
at the end of the tip.
- The tip of the pipette should not touch the bottom side of the well, it
should be just inside the well.
- Use a new tip each time a new sample is add.
- Once you finish loading the samples don’t move the gel tank because
this will cause sample contamination.

23. Load 10 μl from the DNA pre stained ladder.

24. Close the lid of the tank and make sure to attach the electrodes in the
correct positions.

Stage 5: Electrophoresis
25. Set the power supply on 100 volts, this will create electric current across
the gel and will start the separation of DNA fragments.
Notes:
- You will observe bubbles rising from the negative electrode (black
electrode).
- Best resolution is obtained with 4-10 V/cm (cm = distance between the
electrodes, not gel length).
- After few minutes you will observe the dye with DNA fragment migrate
from the wells into the gel.

26. ` Turn OFF the power supply after the bromophenol blue migrates from
the black electrode to near the red electrode then disconnect the
electrodes from the power source.
Stage 6: Visualizing the nucleic acid
27. Place the Gel tray in UV trans illuminator in order to visualize the
separated DNA fragments.

28. Wear a safety goggles then turn off the light.

29. Switch on the UV trans illuminator and you will visualize the separated
DNA Fragments.