Protocol for Gel Electrophoresis:
- obalance/weighing paper
- ograduated cylinder
- oerlhenmeyer flask (small)
- oheat source (hot plate, microwave, etc.)
- ogel box with tray
- omicropipette with tips
- oultraviolet lightbox with integrated camera
- opower supply
- opaper towels
- oagarose powder
- obuffer solution (1X TBE)
- oDNA (standard ladder for this experiment)
- oDNA loading dye
- oethidium bromide staining solution
Part one: Preparing the agarose
1.) To prepare a 1% agarose solution (1% means that for every one gram of agarose powder, add 100mL buffer solution). Use a balance to measure 0.4g of agarose powder on the balance, and put it into the flask.
2.) Measure 40mL of buffer solution in the graduated cylinder, and pour it into the flask. Swirl gently to mix the powder and solution.
3.) Microwave the mixture in the flask until it is just beginning to boil. Do not overboil the mixture. Remove from microwave carefully, using a glove or towel. Swirl gently to mix again and see if all the agarose has melted. If not, microwave a little longer.
4.) Allow the flask to cool down to about 55 degrees Celsius, or until the flask is cool enough to touch.
5.) While the agarose is cooling, prepare your gel tray for the pouring. Insert the gel tray into the gel box, allowing the rubber gaskets to form a seal with the sides of the gel box. Make sure there are no holes through which the melted agarose can leak. Insert the comb into the gel tray.
6.) Using a micropipette, add 4µL ethidium bromide solution to the cool mixture. Swirl gently to mix. Carefully pour the cooled agarose into the gel tray, taking care not to overfill it (a good level for the liquid is about halfway up the comb). Remember, ethidium bromide is a dangerous chemical. Do not spill any of the cooled agarose solution. You should be wearing gloves. Allow the gel to polymerize for about 15 - 20 minutes. Do not move or jar the gel while it is solidifying. The gel will turn from clear to opaque.
7.) When the gel has solidified, remove it from the gel box (wearing gloves) and replace it in the gel box so the wells are in line with one of the electrodes on the box (see photo). Gently fill the gel box with buffer solution, taking care to see that the comb and wells are almost covered by the solution.
8.) Gently remove the comb from the solidified gel. Take care not to tear the wells. The buffer solution should allow ease in removing the comb and prevent ripping of the wells.
Part Two: Loading the Gel
1.) Using a micropipette, add the appropriate amount of DNA loading dye (blue) to your DNA sample. Generally, a little goes a long way. 1µL of loading buffer is plenty for a DNA sample of volume up to about 15µL.
2.) Pipette the mixture into the empty well, taking care not to push the tip through the bottom of the well and puncturing it. Steady the pipette with your other hand as you gently lower it into the well. Dispense the liquid into the well slowly.
3.) Using a new tip, similarly pipette 5µL of DNA standard ladder into an adjacent empty well.
4.) Place the cover onto the gel box, lining up the negative lead cord (black) with the electrode on the top of the box where the wells are, and the positive lead cord (red) with the electrode on the bottom of the box. Gently fit the cover into place.
5.) Now plug the negative and positive lead cords into the corresponding sockets (red and black) on the power supply. See same photo as step 4.
6.) Turn on the power supply and set the voltage to approximately 80 volts. Allow the DNA to migrate through the gel for about 40 minutes to one hour. You can follow the migration of the DNA by actually seeing the blue color in the DNA loading dye move down the gel. NEVER attempt to remove the cover from the gel box, or touch the gel box in any way while the power supply is turned on.
7.) After enough time has elapsed, turn off the power supply, and remove the gel (using gloves), still in its casting tray. Photograph the gel under ultraviolet light.
8.) Dispose of gel in proper toxic waste container when finished.