Gel:

• agarose
• 0.5X TBE
• ethidium bromide

DNA Sample (Digest):

• 10uL DNA digest
• 5uL dye
• DNA Ladders

Making the gel

1. For most procedures, a 1% agarose gel is sufficient. If you need to separate smaller pieces of DNA fragments (<1 kb) you may wish to use a 1.5% or 2% gel. If you wish to separate larger fragments, you may wish to use a 0.8% gel.
2. A 65mL solution for a 1.5% gel requires 0.975g of agarose. Mix this with 65mL of 0.5X TBE buffer in a 125mL erlenmeyer flask.
3. Microwave in order to dissolve the agarose in the buffer. If the buffer is allowed to boil, it with froth out of the flask, so stop the microwave before this happens. Take the flask out after each stop and swirl it to mix. Do this until the agarose is completely dissolved. CAUTION: the agarose turns transparent, and so may appear to be dissolved when it is not. Bring it up to the light to make sure it is homogenous.
4. Add 1uL of ethidium bromide for every 10mL of gel solution. WARNING: Ethidium bromide is mutagenic, so take proper precautions. Wear gloves and goggles. Dispose of the tip in the ethidium bromide trash (yellow trash bag).
5. Allow to cool until you can touch the sides for 4-5 seconds without gloves.

Running the Gel:

1. Place the forming tray into the rig. For the smaller rigs, use red rubber stoppers in order to prevent the gel from spilling out of the tray. In the larger rig, tape the sides as shown below. The open end should face the walls of the rig, so that all four sides are covered. This is the ensure that once the gel is placed into the rig, none will spill out.
2. Pour the gel into the forming tray when it cools. If bubbles appear, use the comb to push them towards the sides.
3. Place the comb at the end of the tray. Cover the tray with aluminum foil. Let the gel cool for 20 minutes until solid.
4. Using both hands, grasp the top of the comb between your thumb and index finger, and slowly pull the comb up. A partner should also gently hold down the gel.
5. Rotate the gel 90 degrees so that the opening of the wells are the furthest from the red wire on the cover. Remove the red tape from the forming tray.
6. Add more 0.5X TBE buffer to the rig until the entire gel is covered by about 1mm.
7. Mix loading dye with DNA solution – 5uL for every 10uL of solution. Generally, each well can hold about 30uL total. If you are only using the gel for evaluation purposes, 10uL solution with 5uL dye is plenty. If you need to gel-extract, you should do about 20uL solution and 10uL dye, and can even increase this and fill multiple wells to increase yield.
8. Load DNA and standards into the wells. Check the concentration of your ladder to determine how much you should dilute it down with nuclease free water. You should load standards on both sides of the gel in case your gel runs unevenly. If you know you will be gel extracting later, skip a well between different DNA samples to make sure that the DNA doesn’t mix.
9. Place the cover on the rig and attach the electrodes. Set the voltage to approximately 100V. Let it run for approximately one hour or until the DNA has run ⅔ along the gel. Don’t let it run too long because the ethidium bromide is positively charged, and will shift locations.