Gel Electrophoresis

Joey Schmitzer
Honors Biology Period 9
May 25, 2016

Introduction:
Restriction enzymes are what cut the DNA molecules. They do this when they
scan a DNA molecule and look for particular sequences. These sequences are
usually made up of four to six nucleotides. They are used often in gene
technology by scientists. This is the process of modifying genes and
transferring them to different organisms. Restriction enzymes can also prove
useful for recombinant DNA technology. Restriction Fragment Length
Polymorphism (RFLP) is the name for a variation in a sequence of DNA. These
are found through gel electrophoresis. RFLP was one of the first techniques
for DNA analysis, which also includes the use of restriction enzymes. Gel
electrophoresis is a method used to separate DNA, RNA, or proteins
according to size. The molecules separates in gel electrophoresis are moved
by an electrical current through a gel containing small pores. In our lab, we
made our own gels and used the gel electrophoresis method. The purpose
was to make our own individual analysis on gel electrophoresis and see how
different restriction enzymes cut DNA. In doing this, we were gaining
experience with restriction enzymes and gel electrophoresis. After
conducting our experiment, we wanted to create a logarithmic graph with
known data to figure out the other lengths of our DNA fragments that were
created by different restriction enzymes cutting them.
In this lab, the independent variable was the different restriction enzymes
used to cut the DNA, while the dependent variable was the lengths of the
DNA fragments after going through gel electrophoresis. The control group, in

this case, will not have a restriction enzyme. The hypothesis agreed upon
was that the different restriction enzymes would cut the DNA into different
sized fragments rather than same sized fragments.

Materials:

Agarose Gel
TBE Buffer Solution
Lambda DNA
Restriction Enzymes (Ecor1,
BamHI, HindIII)
Micropipettes
Micropipette tips
Hot plate
Eppendorf reaction tubes
50 mL beakers
1000 mL flasks

Electrophoresis chamber
Graduated cylinder
Micro centrifuge
Vortex
Ethidium bromide stain
Loading dye
Gloves
Goggles
Staining trays
Ultraviolet light source
Sharpie

Procedures:

1.
2.

3.
4.

A: Set Up Restriction Digest

Label four 1.5 ml tubes in which you will perform restriction reactions: B for BamHI, E
for EcoRI for HindIII, and for no enzyme.
Use table below as a checklist while adding reagents to each reaction. Read down each
column, adding the same reagent to all appropriate tubes; use a fresh tip for each reagent.
All groups share the same BamHI, EcoRI, HindIII enzymes at a central station.
Pool and mix reagents by tapping the tube bottom on lab bench, or with a short pulse in
micro centrifuge.
Incubate all reaction tubes for a minimum of 20 minutes at 37 degrees Celsius. Your
teacher may instruct you to incubate the reactions for longer.

B: Cast Agarose Gel

C: Load Gel

1. Add 1 ul loading dye to each reaction tube. Mix dye with digested DNA by tapping tube
on lab bench, or with a pulse in micro centrifuge.
2. Use micropipette to load contents of each reaction tube into a separate well in gel, aligned
as illustrated in ideal restriction digest of lambda DNA. Use a fresh tip for each reaction
tube.
a. Steady pipet over well using two hands.
b. Be careful to expel any air in micropipette tip end before loading gel. (If air bubble
forms cap over well, DNA/ loading dye will flow into buffer around edges of the
well.)
c. Dip pipet tip through surface of buffer, position it over the well, and slowly expel the
mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the
bottom of the well. Be careful not to punch tip of pipet through bottom of gel.

D: Electrophorese

1. Close top of electrophoresis chamber and connect electrical leads to an approved power
supply, anode to anode (red-red) and cathode to cathode (black-black). Make sure both
electrodes are connected to same channel of power supply.
2. Turn power supply on and set voltage as directed by your instructor. Shortly after current
is applied, loading dye can be seen moving through gel toward the positive pole of
electrophoresis apparatus.
3. The loading dye will eventually resolve into bands of color. The faster the moving,
purplish band is the dye bromophenol blue; the slower-moving, aqua named is xylene
cyanol. Bromophenblue migrates through gel at same rate as DNA fragment
approximately 300 base pairs long. Xylene cyanol migrates at a rate equivalent to
approximately 2000 base pairs.

4. Allow DNA to electrophorese until the bromophenol blue band nears the end of the gel.
Your instructor may monitor the progress of electrophoresis in that case omit steps 5 and
6.
5. Turn off power supply, disconnect leads from inputs, and remove the top of
electrophoresis chamber.
6. Carefully remove casting tray and slide gel into staining tray labeled with your group
name. Take gel to your instructor for staining.

Results:

The picture above shows an ideal gel with DNA fragments. This ideal
gel was used to measure the distance between fragments, which led to
the results shown below.

Distance Travelled by Fragments Cut with HindIII

1.6
1.4
f(x) = - 0.01x + 1.9

1.2
1

Fragments Size (log kbp)

0.8
0.6
0.4
0.2
0
30

40

50

60

70

80

90

100 110 120 130

Distance Travelled by Fragment (mm)

This picture is a graph of the distance travelled by the fragments that

were cut using HindIII. It shows the best-fit line among these data
points and its linear equation.

Distance Travelled and kbp Lengths

HindIII
EcoRI
BamHI
D
A
D
C
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This is a chart that displays the calculated base-pair sizes with the
actual bas-pair sizes. The calculated results were found using the
equation in the previous picture, and then taking the inverse log of that
answer.

Discussion:

The hypothesis that was concluded was that the different restriction
enzymes would separate the DNA into fragments at different lengths
with different sizes. The hypothesis was proven correct when the three

enzymes had three different results. The shorter fragments travelled

further than the longer fragments, and the fragments would group
together by size.

This experiment had several areas where an error could have occurred.
One might measure the distance of the fragments inaccurately, which
would lead to inaccurate results. Another error could occur due to
equipment failure. The gel concentration must remain consistent with a
steady voltage to avoid fragment migration that is too slow or fast. As
well, if the buffer solution is not of the right composition. A buffer with
the wrong pH or ionic concentration will change the shape of the
fragments and their migration times.
References:
DNA Restriction Analysis Lab Manuel
www.learn.genetics.utah.edu/content/labs/gel/
www.nature.com/scitable/definition/gel-electrophoresis-286
www.dnalc.org/resources/animations/gelelectrophoresis.html