DNA GEL ELECTROPHORESIS

By Adam Mejia

Gel electrophoresis is a technique used by biological scientists to separate DNA according to their
fragmented size under the influence of an electrical field through a porous gel. This technique is
commonly used in labs to visually determine the size of DNA sought for in an experiment[1].

Electrophoresis

The event of gel electrophoresis happens when DNA molecules are subject to migration when
they are charged by an electrical field; DNA migrates down through a gel. This happens when
DNA is negatively charged and it migrates because of attraction to a positive anode, which is an
electrode with a positive current of electricity.

Gel

In gel electrophoresis, DNA is separated into bands of fragments of a variety of sizes in base
pairs by a porous gel. Most gels used in gel electrophoresis are made of a sugar compound
called agarose. Agarose is a polysaccharide, or structure of more than one sugar. It is derived
from a marine algae gelatin substance called agar. When an agarose gel is being made it needs
to be made with a solvent, a liquid, called a buffer that will allow the forming of a useable gel.
When agarose is melted in the buffer and cooled, the solution polymerizes. This means that the
substances form a more complex structure, and in this case, agarose forms a micro tangle of
strands. To understand what this looks like, imagine a wad of lint that forms into a matrix when
collected in a dryer lint trap[1][2].

DNA Separation

DNA that is subjected to gel electrophoresis begins as a homogeneous solution, or a solution that
is uniform and doesnt settle into layers like oil and water. The DNA separates according to the
size of base pairs. Base pairs are the single nucleotides/basic structures of DNA called: Adenine,
Guanine, Cytosine, and Thymine. These nucleotides are used as a basic unit to measure DNA
and gene length. The base pair fragments of DNA relate to how far they will migrate down the
gel. Imagine a prospector searching for gold and they have different kinds of sieves to separate
large rocks, stones, pebbles, and gold flake from each other. In the event of gel electrophoresis,
the smallest bands of DNA will be seen to have migrated the farthest because they are able to
move through the gel matrix the easiest.
 Gel Electrophoresis Device and Process
Gel Electrophoresis Device - Horizontal Mini Gel Electrophoresis Systems

The device that actually allows scientists to perform gel electrophoresis is one of the more simple
devices found in a lab. For gel electrophoresis to work, the following essential components need
to be present: gel electrophoresis chamber, a liquid buffer for an electric current to pass through,
the lid that connects charge leads to a power supply, a power supply for constant charge, and the
gel casting tray where the gel is formed for the experiment[3].

Electrophoresis Chamber

The electrophoresis chamber is a plastic box that houses all the materials for gel electrophoresis
to occur. It is small enough to use on a lab bench and measures 22x15x9.5 cm. The chamber in
this description illustrates horizontal gel electrophoresis. Here you will see two color coded prongs
on the side of the chamber for the anode and cathode terminals (positive and negative charges)
where the power supply connects. The anode is color coded red to denote where positive current
flows, and the cathode is color coded black to show where negative current flows. (figure 1)

Figure 1 - The black and red

terminals on the other side of the
chamber are the cathode and anode.
This model of horizontal electrophoresis
device is the Owl EasyCast B1 Mini
Gel Electrophoresis System

The cathode and anode terminal prongs receive power through wire leads that are connected to
the lid. These wire leads connect to a power supply that puts out a current in volts, and can be
programmed to run a time in minutes for electrophoresis.

Gel Buffer

In order for gel electrophoresis to work, a stable liquid called a buffer is poured into the gel cham-
ber to the point where the cathode and anode are covered. The chamber of the featured model
holds a volume of 600 mL. If the chamber is filled properly, then during gel electrophoresis there
should be bubbling on each end of the chamber[4].

Gel Tray

The gel casting tray, also made of plastic, is where the agarose gel is formed into a shape that
fits in the chamber. This tray measures 11x9 cm. The gel and tray sit atop a raised section of the
chamber in the center. (Figure 2) The slots in a gel casting tray are where toothed combs are
placed before the gel is poured. The combs form what are called wells where DNA is loaded for
gel electrophoresis. Usually well combs are 1 mm thick and about 1 cm wide and number from 6
to 10 well forming teeth. (Figure 3)[5]
 Figure 2 - The gel casting tray with gas-
kets with slots for combs

Figure 3 - Gel well forming combs num-

ber from 8 well forming teeth and above
in this figure.

Gel Electrophoresis Process

Sample Preparation

The first step in gel electrophoresis is to prepare DNA samples for loading into the wells. This is
done by adding a loading dye to the DNA, which makes the DNA visible when it travels through
the gel and be able to see individual DNA fragments when subject under UV light. The prepared
samples are measured in microliters (uL) that can range depending on the volume capacity of the
wells. The samples to be loaded can be anywhere from 5 to 34 uL in volume.

Agarose gel

Next, an agarose gel solution is prepared by mixing dry agarose stock with the same kind of liquid
buffer found in the electrophoresis chamber. For example, a common buffer used in gel electro-
phoresis is TAE buffer because it maintains the pH balance of DNA to protect its structure when
the DNA migrates through the gel. When the agarose and buffer are mixed, the solution is heated
until agarose crystals cant be seen in the mixture; a clear solution should be obtained. The solu-
tion is allowed to cool until the container it is in can be handled without discomfort to the hands.
A well forming comb is placed in the gel casting tray, then the tray is sealed with either tape, or
the gaskets of the tray are tightly against the walls of the chamber. Water can be poured in the
tray to ensure there is a seal, then dry the tray. The gel solution is then poured into the tray to
completely cool, and in approximately 20 minutes the gel solution will be a solid gel. Next, the gel
comb is removed and the tray is placed in correct position (the wells of the gel should be closest
to the cathode - the black terminal). The chamber is then filled with TAE or another buffer until the
fill line, or until it completely covers the agarose gel.[6][7]
Sample Loading

Following that, a DNA ladder (a product made by a company that includes specific fragment sizes
of DNA to visually compare measurements of your sample DNA fragments) is usually loaded into
the first well. However many increments of DNA in microliters is then loaded into the next wells.
After the lid is placed on, and most gel electrophoresis lids always orient the power leads to the
correct terminals because of structural design. When the device is set up, the wire leads are
inserted to the power source making sure the color of the wire leads match the color of the sock-
ets. Next, the power supply is programmed to the correct voltage, which could be 120 Volts or
130 Volts. The time for electrophoresis can also be programmed with the power supply, which is
usually programmed from 35 minutes to 50 minutes.

UV Analysis

When gel electrophoresis is complete, the gel is removed from the chamber and placed in a UV
Transiluminator, and this device allows you to see DNA bands illuminated by UV light and see
where bands have migrated. Analysis of the bands in comparison with the DNA ladder fragment
sizes reveal the size of your samples fragments. Most UV booths also allow you to take a picture

Figure 4 - an example of what a

photographed gel looks like under
UV light. DNA bands are labeled
with their base pair size, and wells
are labeled with the sample names.
 Procedure: Gel Electrophoresis[6][7]

In this procedure you will learn how to assemble your gel electrophoresis device, prepare
an agarose gel, prepare your DNA samples, and perform gel electrophoresis.

Materials:

Horizontal Mini Gel Electrophoresis System: buffer chamber, EasyCast gasketed gel tray,
SuperSafeTM lid
6 well gel comb
1x TAE buffer
Stock agarose - dry powder
Analytical scale
Reagent spatula
Weigh boat
100 mL bottle with cap
100 mL graduated cylinder
Ethidium bromide
6x Loading dye or 5x SYBR Green loading dye
Microwave
Lab tape
GeneRuler 1kb DNA Ladder 0.5ug/uL (loading dye added)
Purified DNA samples
P-10 and P-20 micropipettes with tips
Gel electrophoresis power supply
Lab gloves
Oven hot pad/glove
Procedure:

1. Don your disposable lab gloves.

2. Gather the gel electrophoresis system: buffer chamber, gasketed gel tray, and the lid. Make
sure to grab a 6 well gel comb too.
3. Next, you will make a 1% agarose gel. To do this you use a percentage equation, for example
of a 1% solution: 1 g/100 ml= x g/50 ml. Note: gel solutions for horizontal gel electropho-
resis need to total at 50 mL.
A) To find x grams to use you: (1 g x 50 mL)/100 mL = x grams. In this case, x will = 0.5
grams of agarose mixed with 50 mL of buffer. Remember: The solution percent depends on
how many grams of solute on the left side of the equation. For example, 1 g = 1%, or 5 g =
5%.
4. Measure 0.5 g of agarose stock onto a weigh boat using an analytical balance and reagent
spatula.
5. Pour your agarose solute into a 100mL bottle.
6. Next, pour 50 mL of 1x TAE into your bottle and mix by swirling the bottle.
8. The solution is not ready because the agarose crystals are not dissolved, so you need to
melt the crystals by placing the bottle in a microwave for 45 seconds.
9. Afterwards, use a hot pad to grab the bottle and swirl your contents, then check to see if
you can see crystals that look like bits of glass floating around. If there are visible crystals,
then place your mixture in the microwave to melt more for 15 seconds. Repeat for 15
seconds until your solution looks clear, and that will indicate the agarose melted completely.
10. Next, add 1.0 uL of ethidium bromide to the gel solution and swirl to mix. Caution: Before
adding ethidium bromide, make sure youre wearing gloves. Ethidium bromide is a
mutagen, meaning that it has the potential to cause cancer if it gets on you! However,
by using SYBR Green loading dye, you dont need to use ethidium bromide.
11. Allow the mixture to sit aside on your lab bench to cool.
12. As the gel solution is cooling, prepare to seal your gel tray by snuggly fitting the gasket ends
with the inside walls of the electrophoresis chamber. Troubleshooting: If your gel tray does
not have gaskets or has damaged gaskets, then seal the ends with lab tape and add water
to check the seal.
13. Place your 6 well comb into the top slot, not the middle slot, of the gel tray.
14. Your gel solution is ready to pour into form when the bottle can be handled comfortably.
Pour slowly into the gel tray to prevent bubbles from appearing.
15. Immediately rinse out the bottle with hot water to dissolve away left over agarose.
16. Allow your gel to solidify until 20 to 30 minutes.
17. While the gel is solidifying, prepare your DNA samples with loading dye. To know how much
loading dye to use you divide the sample volume by the concentration of the loading dye, for
example: If your DNA sample is 20 uL and your loading dye is 5x/uL concentration then 20
uL/5= 4 uL. 4 uL will then be added to the DNA sample using a P-10 micropipette.
18. Use a vortexer to mix each sample or use a micropipette to mix using a clean micropipette
tip for each sample.
19. When your gel has solidified, carefully remove the gel comb by slowly pulling up, so as to not
damage the gels wells.
20. Carefully remove the tape from both ends of the tray, or if you have the tray sealed by the
gaskets, then pull out the tray and turn the tray with the wells closest to the anode (black
terminal of negative charge). DNA is negatively charged, so you want the DNA to migrate to
the red cathode through the gel and not run out above the wells.
21. Fill the gel electrophoresis with 1x TAE buffer up to a fill line indicated on the box, or ensure
enough buffer is covering the gel. Make sure your gell wells are also filled with buffer.
22. Load 5 uL of your DNA Ladder into your first well using a P-10 micropipette and fresh tip.
Note: It helps to use your other hand to stabilize your working hand to prevent spilling
away from your wells or damaging the wells with the micropipette tip.
23. In your second well and consecutive wells, add your desired amount of DNA samples with
a P-10 or P-20 micropipette. Tip: Add DNA sample to every other well after the DNA
Ladder if youre unsure of spilling over into another well.
24. When your wells are loaded with sample, then attach the SuperSafeTM lid to the
25. Attach the leads that are coming from the lid to the power supply. Make sure youre attach
ing the black lead wire to the black port and the red lead wire to the red port.
26. Turn on the power supply, set the voltage to 120 or 130 volts, and start the power supply.
Important: When the voltage is running, you should be able to see bubbles rise from thin
wires at the bottom of the electrophoresis chamber. This tells you that electrophoresis is
occurring
27. Allow gel electrophoresis to run for 40 to 45 minutes, or until your bands of dye have
traveled halfway through the gel.
28. When gel electrophoresis is finished, turn of the power supply, carefully remove the gel,
and observe your DNA bands under UV light in a UV Transilluminator booth.
When the gel is photographed, it should end up looking like this[9]:

Cassill, J. P., & Ness, B. P. (2015). Gel electrophoresis. Salem Press Encyclopedia Of Health,
1

Alder, R. (2016). Electrophoresis. Salem Press Encyclopedia of Science.

2

3
Thermo Fisher Scientific. 2012. OwlTM B1 EasyCastTM Mini Gel System Illustration.
< https://tools.thermofisher.com/content/sfs/manuals/D21171~.pdf>

4
Thermo Fisher Scientific. March 2017. OwlTM B1 EasyCastTM Mini Gel Electrophoresis.
< https://www.thermofisher.com/order/catalog/product/B1?ICID=search-product>

5
Topac Inc. 2014. Horizontal-Electrophoresis Combs MultiSUB mini system.
< http://topac.com/combs_multSUB.html>

6
Mejia, Adam. (2016). Gel Electrophoresis. Lab Notebook #1. Pg. 20-24

7
Bower, Jean. (2016). Gel Electrophoresis Activity. Intro To Biotechnology Lab. Salt Lake Com-
munity College.

8
Mejia, Adam. (2016). Plasmid Identification. Lab Notebook #1. Pg 37.

9
Thermo Fisher Scientific. March 2017. ReadyPouchTMAgarose Gel.
<https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/DNARNA-
Purification/Images/215/LEFT_1percent-agarose-new.jpg>