Pharm Sci/Chem 177L

Winter 2016

II. Agarose Gel Electrophoresis

Agarose gel electrophoresis is a technique used in molecular biology to
separate nucleic acids by size. An electric field is applied to the agarose gel
and the negatively charged nucleic acids causes them to migrate through
the gel matrix toward the positive electrode based on length, with the
shortest macromolecules running the farthest. A molecular size marker or
ladder is often run alongside the sample to allow for accurate size
determination. Visualization of nucleic acids is achieved by addition of a UV
responsive chemical stain, which reacts with the nucleobases and allows
them to be seen under a UV light source.
Protocol for casting a ____% gel
1. Obtain a 250 mL Erlenmeyer flask and add 40 mL of 0.5 x Tris borate EDTA
buffer (0.5 x TBE). TBE is a buffer used to cast and run agarose gels.
2. Weigh out 0.8 gram of agarose and add it to the 0.5x TBE in the flask. The
easiest way to ensure that you do not get any clumps when adding the
agarose is to swirl the flask to get the buffer moving and then pour in the
agarose powder.
a. What percent gel is this?
b. How much agarose would you need to add to make a 3% gel?
3. Prepare the gel box by placing it on a level surface to ensure that the gel
is cast uniformly. Place the gel boat in the box so that the seals on the boat
are pressed against the rubbery sides of the casting apparatus. This will form
a seal so that the hot gel does not escape before it cools and solidifies.
Alternatively, you can use masking tape to seal the ends of the gel boat for
casting.
4. Place a Kim Wipe in the top of the flask that contains the TBE and agarose.
This helps prevent the solution boiling over when it is heated.
5. Heat in the microwave for 45 seconds, or until boiling. Carefully remove
from the microwave and swirl the solution. Place back in the microwave and
heat for another 20 seconds. The liquid should be clear and all the agarose
dissolved. If not, continue heating in increments of 15 seconds until this is
achieved. Be careful to watch the solution in the microwave. Stop heating
once it boils, or you lose excess volume. The flask is very hot! Take extra
precautions removing from the microwave.
6. Carefully pour the gel into the prepared gel box. Use the comb to smooth
out any air bubbles.
7. Place the comb into the slots at one end of the gel box. This will make the
wells where you will load your samples. Once the gel is poured do not move
the box until the gel is cooled and is solidified. This takes about 20 minutes
and the gel will be slightly opaque.
Protocol for loading and running samples

Pharm Sci/Chem 177L

Winter 2016

1. Obtain a small piece of Parafilm and press into a PCR rack to create one
depression for each sample you need to run on your gel. You will also need one
extra depression for a DNA ladder you will use to size your samples. Pipette
2 L SYBR Gold loading dye into each depression and then add 2 L of each
one of your samples to one depression. Add 2 L of DNA ladder to the final
depression. SYBR Gold is a nucleic acid stain that is used to visualize the
sample under UV light.
The structure of the similar SYBR Green:

The excitation and emission wavelengths for SYBR Gold: 495 ex /

660 em
2. Rotate the gel boat with the prepared agarose gel so that the line of
wells is perpendicular to the long side of the gel box, and closest to the
black electrode.
3. Remove the comb and add enough 0.5x TBE to the gel box to cover the
gel and fill the reservoir on either side. Make sure that there are no air
bubbles in the wells.
a. What charge are nucleic acids and what causes this charge?
a. What charge should the black electrode be to allow for migration of
the nucleic acids into the gel?
4. Mix your first sample and load the gel by pipetting directly into a well.
Make sure to hold the pipette as vertical as possible and with both hands to
minimize shaking. Place the tip in the well, being careful not to puncture the
gel at the bottom of the well. Slowly dispense the sample into the well. The
PCR mix and ladder have density factors included and assures that your
sample stays in the well and does not float out.
5. Change tips and continue loading your samples until they are all loaded,
including a marker ladder (50 bp ladder). Place the cover of the gel box and
connect the color coded electrodes to the power supply. (Black cable gets

Pharm Sci/Chem 177L

Winter 2016

plugged into the black electrode on the power supply and red connects with
red.)
6. Turn on the power supply. Set the voltage to 200 volts and the time for 15
minutes. Press start. You should see small bubbles rising from the electrode
closest to the wells and the loading dyes starting to migrate into the gel.
7. Once the power supply turns off, remove the gel from the gel box and
place it on the UV transilluminator. Turn on the UV light source and collect an
image with the camera provided, or use your own.