Gel Electrophoresis

Matt Koenig
Honors Biology
May 13, 2016
Period 9

Introduction
Gel Electrophoresis is the process of using restriction enzymes to determine patterns in
DNA, usually for the purpose of finding a match between certain samples of DNA. Restriction
enzymes are a class of enzyme that cut DNA molecules when they recognize a repeating pattern.
For example, the restriction enzyme BamH1 cuts DNA wherever recognizes the pattern of base
pairs GGATCC. Because each person has a unique sequence of DNA base pairs, the lengths of
the fragments will only match that persons own DNA fragment lengths. However, often more
than one restriction enzyme must be used to determine a positive match. Scientists use these
restriction enzymes for a range of purposes. They are commonly used for gene technology, or
inserting genes into a genome. They are also utilized for criminal cases in which DNA is left
behind on a crime scene, and can be used to determine paternity cases.
Gel Electrophoresis is how these restriction enzymes can be put to use. Because this
process is commonly used in criminal cases, it will be explained as if forensic scientists were
attempting to find a match to DNA found at a crime scene. When a sample of DNA is collected,
it is preserved and duplicated in order to avoid the chance of the specimen being damaged. It is
then dyed and, along with the other sample(s) of DNA (acquired from suspects) put into the gel.
The gel is a firm substance, but is permeable enough for the fragments of DNA to move through
it. The gel is placed in water. In the first well of the gel is the control group, the untainted
sample of DNA found at the crime scene. The subsequent wells would be filled with the DNA of
suspects. Each sample would have been mixed with the same restriction enzyme. The water is
then charged, the side with the wells negatively, and the side opposite the wells positively. The
DNA fragments will move towards the positive charge, with the smaller fragments moving
further than the larger fragments. When the process is completed, the fragments are measured
and compared. If there is a single match, the suspect is likely guilty. If there are multiple
matches, the process is repeated with a different restriction enzyme. Similarly, if this method
were to be used in a paternity case, if the fragment sizes matched the father could be determined.
There were multiple purposes to the lab. The first purpose was to get practice with
restriction enzymes and gel electrophoresis. The second was to see if different restriction
enzymes cut DNA into different sized fragments or cut them into the same sized fragments. The
third and final reason that this lab was performed was to create a logarithmic graph with known
data to figure out the other lengths of the DNA fragments that were created by different
restriction enzymes cutting them. The hypothesis was that the DNA would be cut into
differently-sized fragments by the different restriction enzymes, while the control group would
remain a single piece.
The original DNA is the control group. The independent variable is the restriction
enzymes added to the DNA. The final sizes of the DNA fragments of the samples with the
restriction enzymes are the dependent variables.

Materials

Agarose Gel
TBE Buffer Solution
Lambda DNA
Restriction Enzymes (EcoRI, BamHI, HindIII)
Micropipettes
Micropipette tips
Hot Plate
Eppindorf reaction tubes
50 mL beakers
1000 mL flask
Electrophoresis chamber
Graduated Cylinder
Microcentrifuge
Vortex
Ethidium Bromide Stain
Loading Dye
Gloves
Goggles
Staining trays
Ultraviolet light source
Sharpie

Procedure
A: Set up Restriction Digest
1. Four 1.5 ml tubes were labeled, in which restriction reactions would be performed: B for
BamHI, E for EcoRI for HindIII, and for no enzyme.
2. Table below was used as a checklist while adding reagents to each reaction. Each column was
read, the same reagent added to all appropriate tubes; a fresh tip was used for each reagent. All
groups shared the same BamHI, EcoRI, HindIII enzymes at a central station.
Tube
DNA
Buffer
BamHI
EcoRI
HindIII
H2O
B
4uL
5uL
1uL
---E
4ul
5uL
-1uL
--H
4uL
5uL
--1uL
--4uL
5uL
---1uL
3. Reagents were mixed and pooled by tapping the tube bottom on lab bench, or with a short
pulse in microcentrifuge.
4. All reaction tubes were incubated for a minimum of 20 minutes at 37 degrees Celsius.
B: Load Gel
1. Premade Agarose Gel was collected
2. 1 uL loading dye was added to each reaction tube. Dye was mixed with digested DNA by
tapping tube on lab bench, or with a pulse in microcentrifuge.
3. Micropipette was used to load contents of each reaction tube into a separate well in gel,
aligned as illustrated in ideal restriction digest of lambda DNA. A fresh tip was used for each
reaction tube.
a. Pipet was steadied over well using two hands.
b. Students took care not to expel any air in micropipet tip end before loading gel. (If air bubble
formed cap over well, DNA/ loading dye would flow into buffer around edges of the well.)
c. Pipet tip dipped through surface of buffer, positioned over the well, and the mixture was
slowly expelled. Sucrose in the loading dye weighed down the sample, causing it to sink to the
bottom of the well. Students took care not to punch tip of pipet through bottom of gel.
C: 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 bromphenal blue; the slower-moving, aqua namd 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

This gel shows the ideal result of the lab. As there was an error with the DNA when the class
performed the lab, this DNA was what was used.

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

1.2
1
0.8
0.6
0.4
0.2
0
30

40

50

60

70

80

90

100

110

120

130

This chart shows the results of the EcoRI enzyme. The equation displayed was used to
determine the kbp of each of the other fragments. The length from the starting point to where the
fragment ended up was measured in millimeters and plugged into the equation.

HindIII
Dis.
(mm)
42.0
46.5
60.5
70.0
83.9
115.5
123.0

Act. bp
27,491
23,130
9,416
6,557
4,361
2,322
2,027

EcoRI
Dis.(mm) Cal. bp
42.0
63.0
71.0
77.0
91.0

21,066
10,861
8,439
6,984
4,491

BamHI

Act. bp

Dis.

Cal. Bp

Act. Bp

21,226
7,421
5,643
4,878
3,530

(mm)
47.0
52.0
64.0
66.0
72.0

17,993
15,367
10,524
9,881
8,177

16,841
12,275
7,233
6,527
5,505

This table displays the calculated kbp of each fragment after being split by a restriction enzyme,
or in the case of the control group, still a single mass.

Discussion
The hypothesis was correct. The DNA was cut into differently-sized fragments by each
restriction enzyme, while the control group remained a single piece. The restriction enzymes cut
the DNA at different locations, which then travelled different distances. The smaller the
fragment, the faster it moved. The sources of error for the group could not be determined, but
they occurred in all other groups that performed the expirement.

References
https://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://biotechlearn.org.nz/themes/dna_lab/gel_electrophoresis
http://www2.le.ac.uk/departments/emfpu/genetics/explained/electrophor
esis