Laboratory Protocols
Agarose gel electrophoresis
Study the principles of agarose gel electrophoresis: in theory and consult the websites
Materials
1. Electrophoresis buffer: 50x concentrated TAE buffer
2. Agarose ultra pure (DNA graded)
3. Agarose gel: 0.7 m/m % in 1x TAE (to 49 volumes agarose solution 1 volume of 50x
TAE is added.
4. Electrophoresis equipment:
electrophoresis tank, gel tray, sample comb and power supply
5. Plastic tape
6. Ethidium bromide: 10 mg /ml stock solution
7. DNA Marker solution:  phage DNA digested with
8. HinDIII gives the following DNA fragments with respect to concentrations:
DNA fragments (in kb):
23.1 9.4 6.7 4.4 2.3 2.0 0.6
Conc. DNA (in ng per band on gel):
952 392 280 184 96 80 16
Method
Notes / questions
1. Ethidium bromide is very dangerous:
Ethidium bromide (2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide), a
mutagen, has a planar molecular structure. It intercalates between the stacked
bases of DNA. Radiation at 300 nm and 360 nm absorbed by the bound dye is
emitted at 590 nm (reddish-orange). There are other possible, non-carcinogenic
dyes that in some cases could replace ethidium bromide: Azure A and B, crystal
violet, Nile-blue-sulphate are the alternatives. See www.ncbe.reading.ac.uk:
DNA revealed. Often these alternative colours are within the visible light
spectrum, so you do not need a UV-light source.
2. The agarose gel should contain TAE or TBE electrophoresis buffer. Why?
3. The DNA has a negative charge when it is in a buffer with a pH of 8.0. Make a drawing
how the power supply cords should be connected to the electrophoresis tank and how
the tray has to be placed and orientated in the electrophoresis tank.
Laboratory Protocols
Preparation of agarose gel
1. Add a pre-weighed mass of agarose to 1x TAE buffer. (Both the buffer selection and gel
concentration may vary). For this experiment a 0.7 w/v % agarose in 1x TAE is prepared in
a Scott flask. Cover the Scott flask and swirl the mixture.
2. Heat the mixture in a microwave to boiling. Continue heating until a clarified solution is
obtained.
3. Remove the hot container using a suitable protective device.
4. Allow to cool to 50-60°C in a pre-warmed water bath
5. Choose an appropriate tray to prepare the right agarose, especially the format of the comb
(with a certain amount of teeth).
6. Tape the ends of a gel tray to prevent leakage of agarose gel from the tray.
7. Pipette with special micropipette 2 l of ethidium bromide solution to the empty tray. Use
gloves whilst pipetting ethidium bromide!
One l of ethidium bromide solution is applied per 50 ml agarose.
8. Dispose of the pipette tip in a container reserved for ethidium bromide waste. Treat the
contents of this vessel with chlorine bleach to destroy the ethidium bromide prior to
disposal.
9. Place the comb onto the tray and pour the agarose gel solution (50-60°C) into the tray.
10. Wait for the gel to harden (about 30 minutes). Remove the tape of the tray and place the
tray in the electrophoresis tank. Keep in mind the charge of the DNA.
11. Fill the tank with 1x TAE electrophoresis buffer. The buffer level should stand 3 mm above
the surface of the agarose gel (so-called “submarine method”)
12. Now remove the comb very carefully, without damaging the sample wells.
13. DNA samples should contain a dye marker such as bromophenol blue (BFB); see
Restriction Enzyme Analysis. This usually contains glycerol to increase the density of the
solution and thereby facilitate loading of samples to the wells.
14. Load samples (26.6 l) into wells on the gel and note in your journal which DNA sample is
pipetted into a certain well.
15. On each agarose gel a DNA reference sample is also applied to two lanes. For a standard
agarose gel, 15 l DNA Marker solution ( phage DNA digested with HinDIII) is used.
16. Cover the electrophoresis tank.
17. Connect the power cords from the power supply to the tank.
18. Turn on the power supply and adjust to a suitable power setting (eg. ca. 80 V constant
charge). Apply power for the desired length of time. (This is usually until the leading edge,
indicated by the migration of the marker dye, moves to the end of the gel.)
19. Turn off the power. Disconnect the power cords, first from the power supply, and then from
the tank.
20. Remove the tank cover and take the tray from the electrophoresis tank.
Transfer the tray to a holding tray without moistening electrophoresis buffer with
ethidium bromide!
21. Wear a face shield to limit exposure to ultraviolet radiation.
22. Place the gel on the UV-transilluminator. Examine the agarose gel for all DNA bands
(reddish-orange) and measure their migration distance in mm. Note these values in your
lab book.
23. Take a photo of the agarose gel; this can be downloaded onto a floppy disk.
24. Dispose of the agarose gel in a container reserved for ethidium bromide waste.
Laboratory Protocols
Questions / tasks
1. Calculate the amount of DNA that is used for the restriction enzyme analysis; what is
the DNA concentration of each of the DNA bands on the agarose gel?
2. Use Excel to calculate the length of each DNA fragment.
For the calibration curve the DNA fragments of DNA Marker solution are used: -phage
DNA digested with HinDIII.
Place the log value of the  phage DNA fragments on the Y-axis and their migration
distance in mm on the X-axis.
From this, calculate the length of the DNA fragments of your samples.
3. Which restriction enzymes have you used? Are the results obtained in accordance with
your expectations?
4. Add a photo of the agarose gel to your lab book.
5. What kind of glass is used in the transilluminator you put the gel on?
Answers to notes/ questions :
Note 2:
If the gel does not contain TAE or TBE, it does not conduct electricity and no separation of
DNA will occur.
Question 5:
Quartz glass: normal glass is not able to let UV light through. On top there is a cover that
does not let UV-light through, in order to protect your eyes.