Team:Waterloo/Notebook/Protocols/Gel Electrophoresis


Revision as of 02:54, 22 October 2009 by Timlin737 (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Gel Electrophoresis

1X TAE buffer
Gel Red
1:10 diluted 1kb DNA ladder
Loading Dye
Gel Rig

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

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

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