Purpose : DNA analysis
Revision date : 2/16/06
Printed on : 2/16/2006
Background : This procedure separates the sizes of DNA usually encountered after restriction. This procedure electrophoreses DNA on a 1% agarose horizontal slab gel. The total amounts of the solutions may vary with the particular gel box, but the ratios of solutions stay the same. Likewise the time of electrophoresis will vary with the gel box.
LE or ME agarose (or similar quality)
50X TAE (Tris-Acetate electrophoresis buffer: 1X is 40 mM Tris-OH, 20 mM Acetic Acid, pH 7.8)
Ethidium bromide (5 mg/ml) [EtBr]
Electrophoresis equipment
Gel loading dye (50 mM EDTA, 0.2% SDS, 50% glycerol, 0.05% w/v bromophenol blue)
Note: The values in parentheses are for the Bio-Rad boxes while the values without parentheses are for the BRL boxes.
1) Prepare 200 ml (300 ml) 1X TAE (Prepared from the 50X stock. In some cases, we have a 10X stock of TAE, so read the label ).
Note: Failure to dilute the TAE will result in very slow migration of the samples and very high amperage. [for the gel that is a little larger, make up 300 ml of buffer]
2) For a 1% gel, add 0.3 g (0.25 g) agarose to 30 ml (25 ml) 1x TAE. To change the percent agarose, adjust the ratio. A 1% gel is 1% weight/volume (w/v). [for example, for the larger gel, make use 0.5 g agarose in 50 ml 1X TAE; for a 1.2% gel, add 0.36 g agarose to 30 ml final volume]
3) Heat the solution to boiling in the microwave to dissolve the agarose. Note: You should not see any beads in the solution. If you have beads in the agarose, you will get distorted bands. For 30 ml, it should take about 50 seconds at power level 10. To reach power level 10, you enter a 0.
4) Add 6 µ l of ethidium bromide to the dissolved agarose and mix. [5 µ l for a 25 ml gel].
Note : Ethidium bromide stains DNA by intercalating between the bases of DNA. It will also intercalate into human DNA, so wear gloves to prevent contact with it.
5) Put the two dams into the slots on each side of the gel plate. Make sure that they fit tight. Pour the melted agarose onto the gel plate in the eletrophoresis box. Next, place the comb (well-maker) in place. Use the comb with the larger teeth if you have 8 or fewer samples, or the small comb if you have up to 14 samples. [If there are no dams, you place tape across the ends of the gel instead of using the dams]
Agarose gel electrophoresis 1

Note: Make sure that the comb is nearest to the black electrode (cathode), as the DNA migrates towards the red electrode (anode).
6) Let the gel cool to room temperature. It should look cloudy, and, if you touch it, the gel will feel cool or cold.
Note: Do not pull the comb out too soon, as it causes the wells to collapse. It will take 15 to 20 minutes to gel.
7) Carefully remove the dams and the comb. Pour in 150 ml (250 ml) of 1X TAE (electrophoresis buffer). Do not pour in too much buffer, as it comes in contact with the electrical contacts. The more buffer in the chamber, the higher the current will be when the gel is run.
8) For the large wells, prepare 10 µ l samples. For the smaller wells, prepare 7 µ l samples. Next, add 3
µ l of the loading dye to your samples. Carefully place the samples into adjacent wells by using an
Eppendorf pipette and a steady hand.
9) Electrophorese the samples at 100 V (80 V) for 45 minutes (30 minutes) or until the dye has migrated at least 6.0 cm.
Note : Check the gel while it is running to make sure it is not getting too hot, as this will distort the bands or melt the agarose.
10) Wearing gloves (since ethidium bromide is present), carefully remove the gel from the box, put it onto the UV light box and take a look. If the gel looks Ok, take a picture.
Note: Wear UV protective glasses or cover the light box with the UV protective shield when the UV light is on. The UV can cause skin cancer.
Problem solving
Problem Solution(s)
Gel doesn’t solidify 1) Not enough agarose added.
2) Not heated enough to dissolve the agarose.
Collapsed wells
Skinny gel or irregular gel
Gel wasn’t cool when the comb was removed
Agarose leaked past the dams. Put the dams in securely.
Well too small (sample won’t fit in the well)
Gel runs too slowly
Gel runs too quickly
Only the ladder is visible under UV light
No bands appear under UV light
Agarose leaked past the dams. Put the dams in securely.
50X TAE not diluted correctly
Little or no 50X TAE added to electorphoresis buffer.
1) Poor DNA purification (e.g., minipreps).
2) Sample leaked out of the well.
1) No ethidium bromide was added.
2) Samples leaked out of the well.
Agarose gel electrophoresis 2

Problem Solution(s)
Fuzzy bands 1) Agarose wasn’t dissolved completely
2) Too much DNA
3) Particulates in the sample
Ladder DNA
(Ladder from Invitrogen, the figure from the manual)
These ladders are from New England Biolabs, and the pictures come from their web site.
The ladder on the left is their 1 kb ladder, while the one on the right is their 100 bp ladder.
Agarose gel electrophoresis 3