The DNA fragments that you will be trying to separate when you run agarose gels in this laboratory will, for the most part, be relatively large (1-6 kb). Therefore most of the gels that you pour will be 0.08% agarose. A higher concentration of agarose is more efficient in separating smaller fragments. A lower concentration of agarose is used when you want to separate larger fragments. The buffer that we will use in this laboratory is 1X TAE. This buffer will be available in the laboratory as a 50X stock solution.

To prepare a gel, measure out the appropriate amount of agarose (Remember this is agarose, not agar). The small gel boxes require 30 ml of 0.8% agarose in 1X TAE; the larger boxes hold 100 ml.¹ So if you were making a 0.8% agarose gel, you would use 100 ml of 1X TAE and 0.8 g of agarose. Place the TAE and agarose in the microwave and heat it on high power for about 2 minutes (until it boils and all of the agarose has gone into solution). Allow the agarose to cool. Set up the gel apparatus in the gel-casting conformation and place the gel comb in the slot provided. Pour the melted gel into the gel form and allow it to harden. The gel will turn slightly opaque when it is has hardened. Turn the gel form to the gel-running conformation, fill the gel box with 1X TAE and slowly remove the gel comb.

The amount of DNA that you load into a well will be different for different applications. In all cases, however, you will add a 1:10 dilution of loading buffer to the sample before loading. Loading buffer contains Ficoll which has a high specific gravity and will "sink" the sample. It also contains two dyes: Bromophenol blue, which runs in a 0.8% agarose gel at the same speed as 4-5 kb linear fragment of DNA, and Xylene cyanol, which co-migrates with DNA of about 500 bp. These guidelines change with the agarose concentration of the gel.

You will always need to run a lane of DNA ladder with your gels. You need only 1 µl of DNA ladder. I always prepare the DNA ladder such that each step of the ladder has about 50 ng of DNA.

When pipeting the sample into the wells, point the pipet tip straight down into the well. Do not try to add the sample at an angle (as most students do); this causes turbulence which can wash the sample out of the well.

Attach the electrodes to the gel box so that the positive electrode is at the bottom of the gel. Why? You will run constant voltage gels and the number of volts that you pass through the gel varies greatly according to the size of the gel and the speed at which you want the gel to run. Typically we will run the small gels at 80-90 V; the large gels usually run at 100-120 volts. The slower you run a gel, the better the resolution of the bands. Run times for gels range from 1/2 hour to overnight depending on the application. Specific instructions will be given with each protocol.

After the gel has run you will place it in a solution of ethidium bromide for about 10 minutes.

ETHIDIUM BROMIDE IS A KNOWN CARCINOGEN AND MUST BE HANDLED WITH EXTREME CARE. YOU MUST WEAR GLOVES WHEN HANDLING ETHIDIUM BROMIDE.

After staining, soak the gel for an additional 5 minutes in water (tap water is fine) to remove excess ethidium bromide from the gel. Since ethidium bromide intercalates between bases in DNA and RNA, and fluoresces under UV light, you can now visualize your DNA in the gel if you look at it under UV light.

Place your gel on the UV light box, turn on the UV and take a picture of your gel. To avoid burns, analyze the DNA from the picture not directly from the gel sitting on the UV light box.

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