Agarose gel electrophoresis protocol
You will be using this technique a couple of times in the course.
Step 1: Pouring an agarose gel
1.) Measure out 35 ml of Tris Borate buffer (TBE) in a 50 ml graduated cylinder and pour into a 125 ml erlymeyer flask. Add the appropriate amount of agarose for the specified percentage gel. In the case of a 1% agarose gel you would add 350 mg of Agarose.
2.) We will melt the agarose using microwave. Place the erlmeyer flask in the microwave and press “Time” key to set time for 1min. After 1 min of boiling take the erlmeyer flask out with a gloved hand and gently swirl the solution. Put the flask back in the microwave and repeat the process to boil for another 1 min or until agarose has fully dissolved. Watch the agarose dissolve; when all has dissolved set the flask down on the bench for 5 minutes.
3.) Taping the gel mold tray. Using masking tape, tape the ends of the gel tray. A gel tray is shown below untaped. Below this is a taped gel tray. Note that the tape is folded around the ends of the gel tray. This stops the Agarose from leaking out the ends.
4.) Pouring the gel. Place the comb 1-2 cm from the top end. Pour the agarose TBE solution into the gel mold. Let the agarose set for 40-60 minutes.
Step 2: Loading an agarose gel
1.) Gently remove the comb. Place the gel mold tray into the running apparatus. Make sure the comb side of the gel is next to the black electrode. Add enough TBE running buffer until the gel is just submerged.
2.) Pipette your samples carefully into the wells.
3.) Place the lid on top. Make sure that the black electrode is back and the red electrode is ahead.
Step 3: Running the agarose gel
1.) Attach the electrodes of the power supply to the gel apparatus. Black on Black and Red on Red.
2.) Turn the power supply ON. There is a switch at the back.
3.) Select Volts and set the power supply to 100 volts.
4.) Hit the run button on the right hand side.
5.) Run the gel for 1 hour
Step 4: Staining the gel
YOU MUST BE WEARING GLOVES FOR THIS!!
1.) Stop the power supply by pushing the run button
2.) remove the lid of the gel apparatus
3.) remove the gel tray and gently slip the gel out into the Ethidium bromide bath. Leave in the bath for 10 minutes.