Agarose Gel Electrophoresis (AGE)

Introduction

Why Electrophoresis?

Introduction of Agarose Gel Electrophoresis

Principle of Electrophoresis

Materials Needed

Methods for Electrophoresis

Application of AGE

Advantage of AGE

Disadvantage of AGe

Assessment

****

A written report prepared by:

NENITA MARIE Q. ALONZO

MaEd General Science

Introduction

Gel electrophoresis is the standard laboratory procedure for separating DNA by size (e.g. length
in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move
the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter
DNA fragments migrate through the gel more quickly than longer ones. Thus, you can determine
the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA
ladder (a collection of DNA fragments of known lengths).

Why Electrophoresis?

• To separate DNA molecules from each other.

• To determine the size of DNA fragments.

• To determine the presence or amount of DNA.

Introduction of Agarose Gel Electrophoresis

• Agarose gel electrophoresis is a method to separate DNA or RNA molecules by size.

• This is achieved by moving negatively charge nucleic acid molecules through an agarose
matrix with electric field (electrophoresis)

• Short molecules move faster and migrate faster than longer ones.

Principle of Electrophoresis

• Powerful separation method of frequently used to analyze DNA fragments generated by

restriction enzymes.

• Convenient analytical method for determining the size of DNA molecules in the range of
500 to 30, 000 base pairs
 • Employs electromotive force to move molecules through a porous gel.

• Separates molecules from each other on the basis of

• -size and/or

• -charge and/or

• -shape

• Basis of separation depends on how the sample and gel are prepared

Figure 1

Materials needed:
 • Electrophoresis chamber

• Agarose gel

• Gel casting tray

• Buffer

• Staining agent (Ethidium Bromide)

• A comb

• DNA ladder

• Samples to be separate

What is Agarose?

• A linear carbohydrate polymer extracted from seaweed, agarobiose

• Forms a porous matrix as it gels

• shift from random coil in solution to structure in which chains are bundled into double
helices.

Figure 2

Gel Casting Trays

Available in a variety of size and composed of UV-transparent plastic.

The open ends of the trays are closed with tape while the gel is being cast, then remove prior to
electrophoresis.

Figure 3

Buffers

Buffers act : to establish pH, and 2) to provide ions to support conductivity. In DNA

electrophoresis, the TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the usual
buffers of choice.

Staining of DNA

• To make DNA fragments visible after electrophoresis, the DNA must be stained.

• The favorite- ethidium bromide

• When bound to DNA it fluoresces under ultraviolet light (reddish-orange color)

Ethidium bromide

• The standard concentration used in staining DNA in gel is 0.5- ug/mL

• Ethidium bromide is a fluorescent between bases of nucleic acids and allows very
convenient detection of DNA fragments in gel.

• Inserting itself between the base pairs in the double helix.

 Figure 4

Comb

• A comb is placed in the liquid agarose after it has been poured

• Removing the comb from the hardened gel produces a series of well used to load the
DNA.

Figure 5

DNA ladder

• is a set of standards that are used to identify the approximate size of a molecule run on

a gel during electrophoresis, using the principle that molecular weight is inversely
proportional to migration rate through a gel matrix. Therefore, when used in gel
electrophoresis, markers effectively provide a logarithmic scale by which to estimate the
size of the other fragments (providing the fragment sizes of the marker are known).

• It is a solution of DNA molecules of different length.

• DNA ladder consist of known DNA size used to determine the size of DNA of an
unknown DNA sample.

The DNA ladder usually contains regularly spaced sized samples which when run on an
agarose gel look like a ladder
 Methods of Electrophoresis

1. Preparation of the Gel

1. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are
prepared using a w/v percentage solution. The concentration of agarose in a gel will
depend on the sizes of the DNA fragments to be separated, with most gels ranging
between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the
capacity of the flask.

2. Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel
running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-
borate, 1 mM EDTA).

3. Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave,
but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl
the contents to mix well. Repeat until the agarose has completely dissolved.

4. Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel may
also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-
30 min, followed by destaining in running buffer for an equal length of time.

Note: EtBr is a suspected carcinogen and must be properly disposed of per institution
regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes
for the staining of DNA are available; however, EtBr remains the most popular one due to its
sensitivity and cost.

1. Allow the agarose to cool either on the benchtop or by incubation in a 65 °C water bath.
Failure to do so will warp the gel tray.

2. Place the gel tray into the casting apparatus. Alternatively, one may also tape the open
edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create
the wells.
 3. Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature.
Remove the comb and place the gel in the gel box. Alternatively, the gel can also be
wrapped in plastic wrap and stored at 4 °C until use (Fig. 6).

2. Setting up of Gel Apparatus and Separation of DNA Fragments

1. Add loading dye to the DNA samples to be separated (Fig. 7). Gel loading dye is
typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30%
glycerol). Loading dye helps to track how far your DNA sample has traveled, and also
allows the sample to sink into the gel.

2. Program the power supply to desired voltage (1-5V/cm between electrodes).

3. Add enough running buffer to cover the surface of the gel. It is important to use the same
running buffer as the one used to prepare the gel.

4. Attach the leads of the gel box to the power supply. Turn on the power supply and verify
that both gel box and power supply are working.

5. Remove the lid. Slowly and carefully load the DNA sample(s) into the gel (Fig. 8). An
appropriate DNA size marker should always be loaded along with experimental samples.

6. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than
the anode (red leads). Double check that the electrodes are plugged into the correct slots
in the power supply.

7. Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

3. Observing Separated DNA fragments

1. When electrophoresis has completed, turn off the power supply and remove the lid of the
gel box.

2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place
the gel tray on paper towels to absorb any extra running buffer.
 3. Remove the gel from the gel tray and expose the gel to uv light. This is most commonly
done using a gel documentation system (Fig. 9). DNA bands should show up as orange
fluorescent bands. Take a picture of the gel (Fig. 10).

4. Properly dispose of the gel and running buffer per institution regulations.

4. Representative Results

Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. After
separation, the resulting DNA fragments are visible as clearly defined bands. The DNA standard
or ladder should be separated to a degree that allows for the useful determination of the sizes of
sample bands. In the example shown, DNA fragments of 765 bp, 880 bp and 1022 bp are
separated on a 1.5% agarose gel along with a 2-log DNA ladder.

Figure 6. A solidified agarose gel after removal of the comb.

 Figure 7. A student adding loading dye to her DNA samples.

Figure 8. A student loading the DNA sample into a well in the gel.
 Figure 9. An example of a gel documentation system.

Figure 10. An image of a gel post electrophoresis. EtBr was added to the gel before
electrophoresis to a final concentration of 0.5 μg/ml, followed by separation at 100 V for 1 hour.
The gel was exposed to UV light and the picture taken with a gel documentation system.
 Figure 11
Applications of AGE

• DNA fingerprinting
• Forensics
• DNA sequencing

Advantage of AGE
• Cheap
• Quick
• Easy
• Sample Recovery

Disadvantage of AGE

• Toxicity
DNA visualization uses dyes like Ethidium Bromide which chelate DNA and is
known as carcinogen. Although some safe alternative is available (SYBR Safe)
Assessment
1. Electrophoresis is used to:
a. Separate DNA fragments
b. Block DNA fragments
c. Purify DNA fragments
d. Both A and C
2. DNA possesses:R
a. No charge
b. Positive charge
c. Negative charge
d. Both charge
3. What is Ethidium Bromide?
a. Buffer
b. Dye
c. DNA solution
d. Restriction enzyme
4. Why do scientists load DNA ladder into the agarose gel?
a. It makes it easier to determine the sizes of unknown using comparison techniques
b. To fill in all the slots so that you can run it.
c. To practice loading the DNA before you get the important DNA.
d. So you know how long the gel runs
5. How can one tell if their gel electrophoresis is running properly?
a. It bubbles
b. You can see the methyl blue move from the well into the gel.
c. The DNA runs to red (anode)
d. All of the above.
6. What is a DNA ladder?
a. it is a marker for the voltage
 b. is a set of standards that are used to identify the approximate size of a molecule
c. it is DNA sample with positive charge
d. all of the above

7. Why do we need to stain the DNA?

a. for visualization
b. for binding of bands
c. for the pH
d. for the fragments to be separated

8. Why do we need to use buffer?

a. for the continuity of separating the bands
b. for the formation of binds
c. to provide ions to support conductivity
d. to provide ions to support the binding

9. Where can we apply Agarose Gel Electrophoresis?

a. DNA fingerprinting
b. DNA sequencing
c. Forensics
d. all of the above

10. Which is a true statement about agarose gel electrophoresis?

a. short bands migrate faster that long bands
b. long bands migrat faster than shortest bands
c. short and long bands migrate at the same time
d. none of the above
 II. Look for the correct procedure of AGE and write in chronological order.

 Add running buffer, load samples and marker.

 Unload the buffer and view the result.
 Stop running the gel when there is no bubbles appear.
 Pour the solution into casting tray and allow to melt.
 View DNA on UV light box and show results.
 Pour the solution into casting tray and allow to solidify.
 Prepare agarose gel.
 Run gel at a constant voltage until band separation occurs.
 Use micropipette to load the agarose gel in the well.
 Runs gel towards cathode.

ANSWER KEY
I.
1. D 6. B
2. C 7. A
3. B 8. C
4. A 9. D
5. D 10. A

II.
1. Prepare agarose gel.
2. Pour the solution into casting tray and allow to solidify.
3. Add running buffer, load samples and marker.
4. Run gel at a constant voltage until band separation occurs.
5. View DNA on UV light box and show results.
References
• https://www.slideshare.net/anamsiddique2/anam-siddique-gel-electrophoresis?
from_action=save&fbclid=IwAR1_XGC3w5IFvpaKKoyUE1rKx7y5fA2ccpiP8Ewa
dD8rPEVLK33dBXqBcS0
• https://biologydictionary.net/gel-electrophoresis/
• http://www.nslc.wustl.edu/courses/bio2960/labs/07DNA/Gel/index.html
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/
• https://www.addgene.org/protocols/gel-electrophoresis/