Team:Waterloo/Notebook/Protocols/Gel Electrophoresis

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<b><u><big>Gel Electrophoresis</big></u></b><br>  

<b>Materials </b><br>
Agarose <br>
1X TAE buffer <br>
Gel Red<br>
1:10 diluted 1kb DNA ladder<br>
Loading Dye<br>
Gel Rig<br>

<b>Instructions for 0.8% Agarose 100 mL gel</b><br>
<u>Prepare the Gel </u><br>
1.  Measure 0.8 g of agarose on weigh paper. <br>
2.  Measure 100mL of TAE buffer using a graduated cylinder into a flask. <br>
3.  Add agarose to flask. Swirl to dissolve as much as possible. <br>
4.  Microwave for 20 seconds, swirl. Repeat until agarose is dissolved. Allow argrose to cool down to room temperature.<br>
5.  Add 2 µL of Gel Red into the gel.<br>
6.  Cover the ends of the gel tray with masking tape to seal them. <br>
7.  Pour the gel into the tray. <br>
8.  Drop the combs into the slots to form the wells. <br>
9.  Allow the gel to set. <br>
10. Remove the combs and rinse with distilled water. <br>

<u>Preparing samples </u><br>
1. Pipette 10µL each digested sample into a labeled microfuge tube (for gel extraction use 20 µL). <br>
2. Add 2µL of loading dye to each sample (for gel extraction use 4 µL) <br>
3. Find the diluted 1kb DNA ladder. <br>
4. Organise the tubes in loading order.<br>
5. Record loading order <br>

<u>Running the Gel </u><br>
1. Place the tray in the rig with the wells closest to the far side. <br>
2. Add TAE buffer until the surface of the gel is just covered. <br>
3. Pipette 10 µL of the digestion mixture and diluted ladders into the appropriate wells.<br>
4. Put the cover on. Black at the back and red at the head. <br>
5. Connect the wires to an available power supply. <br>
6. Set the power supply to 120V and start it. <br>
7. Check that bubbles are forming near the electrodes. <br>
8. Wait about 45 minutes. The dye front should be 3/4 of the way down the gel.<br>