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Team:Northwestern/electrogel
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Revision as of 00:06, 28 September 2013
Electrophresis
- 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.
- Pour the gel into the forming tray when it cools. If bubbles appear, use the comb to push them towards the sides.
- Place the comb at the end of the tray. Cover the tray with aluminum foil. Let the gel cool for 20 minutes until solid.
- 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.
- 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
- Add more 0.5X TBE buffer to the rig until the entire gel is covered by about 1mm.
- 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.
- 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.
- 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.
