Agarose gel electrophoresis

Purpose: DNA analysis Revision date: 4/7/08 Printed on: 4/7/2008 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.

Materials
LE or ME agarose (or similar quality) 10X TBE (Tris-Borate electrophoresis buffer) 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)

Procedure
1) Prepare 1X TBE (Prepared from the 10X stock). This means that for every ml of 10X, you add 9 ml of deionized water. You need about 300 ml per gel run, but we usually make up 2 liters of it. Note: Failure to dilute the TBE will result in very slow migration of the samples and very high amperage (causing excessive heating of the gel). 2) For a 1% gel, add 0.3 g agarose to 30 ml 1x TBE. Note: For analyzing smaller DNA (500 bp or less), use a 1.5% gel. To do this, use proportionately more agarose. A 1% gel is 1% weight/volume (w/v). [for example, for a 1.5% gel, add 0.45 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, then the time. For a 1.5% gel, 70 seconds works better. 4) Add 5 l of ethidium bromide to the dissolved agarose and mix. 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) Get a gel plate and a comb. Put the two dams into the slots on each side of the gel plate. Make sure that they fit tight. They have an angled and a vertical side. Make sure that these match the gel box (vertical side goes inside). Pour the melted agarose onto the gel plate in the

Agarose gel electrophoresis

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 15 samples. 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. This will take 20 minutes. 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. 7) Carefully remove the dams and the comb. Pour in about 250 ml of 1X TBE (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. The buffer must cover the gel to prevent overheating of the gel. 8) If your DNA of interest is < 1 kb: Add 5 l of the 100 bp ladder to the first well (it already contains dye). If your DNA is 1 kb: Mix 0.5 l of the 1 kb plus ladder, 5 l of water, and 3 l of loading dye. Add the whole 8.5 l to the first well. 9) For the large wells, use up to 10 l of your samples. For the smaller wells, use 8 l samples. If there is no dye in your samples, add 3 l of the loading dye before putting it into the wells. Carefully place the samples into adjacent wells by using a pipette and a steady hand. 10) Electrophorese the samples at 100 V for 30 minutes. 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.

Agarose gel electrophoresis

Appendix
Problem Gel doesnt solidify Collapsed wells Skinny gel or irregular gel Well too small (sample wont 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 Fuzzy bands Solution(s) 1) Not enough agarose added. 2) Not heated enough to dissolve the agarose. Gel wasnt cool when the comb was removed Agarose leaked past the dams. Put the dams in securely. Agarose leaked past the dams. Put the dams in securely. 10X TBE not diluted correctly Little or no 10X TBE added to electrophoresis 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. 3) No ladder was used 1) Agarose wasnt dissolved completely 2) Too much DNA 3) Particulates in the sample

Ladder DNA
Bioneer ladders (below). On the left is the 100 bp ladder, on the right is the 100 bp plus (from their web site).

Agarose gel electrophoresis

These ladders are from Invitrogen, and the picture comes from their manual. The figure on the left is the 1 kb plus ladder, while the one on the right is the 1 kb ladder.

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