Gel Electrophoresis Lab

Isabella Haberstock
Honors Biology
May 20, 2016
Pd. 3

Introduction
In this lab, we used three different restriction enzymes on lambda DNA to see how each
would cut up the DNA into fragments. Restriction enzymes are enzymes that each only work for
a certain DNA sequence, which is called a recognition site (5). Once the enzyme finds its
sequence in the DNA, it cuts the DNA in between two nucleotides to create fragments.
Depending on where the certain sequences are, the DNA fragments can be many different sizes
(4). Restriction enzymes are found in bacteria, which uses the enzymes to kill viruses (5).
Restriction enzymes are one of the foundations for biotechnology research. Scientists can
use them to clone DNA, create a DNA fingerprint, present as forensic evidence, and help in
paternity cases (5). Scientists can also use restriction enzymes to cut a DNA sequence in a certain
place so they can insert a foreign DNA fragment into the sequence. An example of this is that
scientists can introduce a new DNA sequence into an E-coli bacterias genome and produce
insulin proteins, which are then used to help people with diabetes. A genome is the part of an
organisms DNA that holds instructions that make every part of a growing being function. Taking
advantage of restriction enzymes and certain genes is called gene technology, and making insulin
is one of many examples (3).
As for paternity and criminal cases, a method of identification called RFLP (restriction
fragment length polymorphism) is used. RFLP is the difference in DNA fragments when the
DNA has been cut by restriction enzymes. Comparing the crime scene sample DNA fingerprint
and those of any possible suspects lead a scientist to who committed a crime. This same method
is also used to find the father of a baby (6).
One way to use RFLP is with gel electrophoresis. Gel electrophoresis is using an
electrical current to sort DNA fragments by size. One end of a gel has many wells in it. Scientists

insert DNA combined with a restriction enzyme into these wells. DNA is negatively charged, so
a negative charge should be applied on the side of the gel containing DNA. The other side of the
gel is positively charged. The DNA will naturally be attracted to the positive and will move
through the gel. The restriction enzymes cut the DNA into different size fragments, and the
shorter pieces will move through the gel faster than the longer pieces. The banded pattern created
by the pieces are a DNA fingerprint. These can be used for various types of identification (1).
The purpose of this lab is to find out if different restriction enzymes will cut the same
type of DNA into the same size fragments or different size fragments. This lab is also to get
practice doing gel electrophoresis and to become familiar with restriction enzymes. The lab also
required a logarithmic graph that was used to find information on the DNA fingerprints, which is
practice for using given information to obtain the rest of the information.
The dependent variable in this lab is the DNA fingerprints created by the banded patterns.
The independent variable is the restriction enzymes. The control group in this experiment is the
test tube containing the lambda DNA with water.
If we use three different restriction enzymes to cut lambda DNA, then each enzyme will
cut the DNA into different size fragments
Materials

Four 1.5 mL tubes

Restriction enzymes: BamHI, EcoRI, HindIII
Water for control tube
Hot water bath
Micropipette
Lambda DNA
Tris-borate-EDTA buffer
Microcentrifuge
Loading dye
Gel casting tray and well-forming comb
Tape

Agarose
Electrophoresis box
Power supply

Procedures
1. Label four 1.5-mL tubes, in which you will perform restriction reactions: B for BamHI, E
for EcoRI, H for HindIII, and for no enzyme
2. Use the table below as a checklist while adding reagents to each reaction. Read down
each column, adding the same reagent to all the appropriate tubes; use a fresh tip for each
reagent. All groups share the same BamHI, EcoRI, HindIII enzymes at a central station.
Tube
B
E
H

DNA
4L
4L
4L
4L

Buffer
5L
5L
5L
5L

BamHI
1L

EcoRI

1L

HindIII

1L

H20

1L

3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a short pulse in a
microcentrifuge.
4. Incubate all reaction tubes for a minimum of 20 mins at 37C.
5. Seal ends of gel casting tray with tape, and insert well-forming comb. Place gel casting
tray out of the way on lab bench, so that agarose poured in the next step can set
undisturbed.
6. Carefully pour enough agarose solution into casting tray to fill the depth of about 5mm.
Gel should cover only about 1/3 the height of comb teeth. Use a pipet tip or toothpick to
move large bubbles or solid debris to sides or end of tray, when gel is still liquid.
7. Gel will become cloudy as it solidifies (about 10 mins). Do not move or jar casting tray
while agarose is solidifying.
8. When agarose has set, unseal ends of casting tray. Place tray on platform of gel box, so
that comb is at negative (black) end.
9. Fill box with tris-borate-EDTA (TBE) buffer, to a level that just covers entire surface of
gel.
10. Gently remove comb, taking care not to rip wells.

11. Make certain that sample wells left by comb are completely submerged. If dimples are
noticed around wells, slowly add buffer until they disappear.
12. The gel is now ready to load with DNA.
13. Add 1L loading dye to each reaction tube. Mix dye with digested DNA by tapping tube
on lab bench, or with a pulse in microcentrifuge.
14. 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 (F3). 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 wells.)
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.
15. Close top of electrophoresis chamber and connect electrical leads to an approved power
supply, anode to anode (red to red) and cathode to cathode (black to black). Make sure
both electrodes are connected to same channel of power supply.
16. Turn power supply on and set voltage as directed by your instructor. Shortly after current
is applied, loading dye can be seen moving toward gel toward positive pole of
electrophoresis apparatus.
17. The loading dye will eventually resolve into two bands of color. The faster-moving,
purplish band is the dye bromophenol blue; the slower-moving, aqua band is xylene
cyanol. Bromophenol blue migrates through gel at same rate as a DNA fragment
approximately 300 base pairs long. Xylene cyanol migrates at a rate equivalent to
approximately 2000 base pairs.

18. Allow the DNA to electrophorese until the bromophenol blue band nears the end of the
gel. Your instructor may monitor the progress of electrophoresis in your absence; in that
case, omit steps 19 and 20.
19. Turn off power supply, disconnect leads from the input, and remove top of
electrophoresis chamber.
20. Carefully remove casting tray and slide gel into staining tray labeled with your group
name. Take gel to your instructor for staining (2).
Results
The lab manual contained a picture of what our gel should have looked like (F1). All
distance measurements were taken from the ideal gel picture. It contains all the correct DNA
fingerprints for HindIII, EcoRI, BamHI, and the control group. Our gel did not resemble the ideal
gel picture and did not have any DNA fingerprints present.
Ideal Gel and Lab Gel

F1. This is a picture of what the ideal gel would look like (left) and our gel (right). The ideal gel shows
how far the DNA fragments moved for each restriction enzyme and the control group. This image shows
the true DNA fingerprint for each enzyme. The actual gel that resulted from this lab shows no DNA
fingerprints.

Using the ideal gel picture above (F1), we took measurements in millimeters from the top
of the gel to the top of each band for the HindIII enzyme. The kilo base pairs for HindIII were
included in the lab manual, so there was enough information to complete the graph below (F2).
We used the information that we had about HindIII to get this graph. After plotting the points, we
inserted a best fit line to get a linear equation that would help us figure out the information for
the other two restriction enzymes.

log of kilo base pairs by distance in mm

1.6
1.4
1.2

f(x) = - 0.01x + 1.82

log of base pairs

0.8
0.6
0.4
0.2
0
30

40

50

60

70

80

90

100

110

120

130

distance in mm
F2. This graph is a scatter plot containing the distance (mm) from the top of the gel to each band and the
log of the kilo base pairs. The individual points on the scatter plot are for the HindIII restriction enzyme,
and a best fit line was added to the graph to estimate the kilo base pairs of the other two restriction
enzymes.

After obtaining the linear equation from the graph above (F2), we measured the distances
for the other two restriction enzymes using the ideal gel picture (F1). We plugged the distance

values in for x in the equation to obtain the calculated kilo base pairs recorded in the table below
(F3). The actual number of kilo base pairs were provided by the teacher.
Distance and Kilo Base Pairs for Each Restriction Enzyme
HindIII

37

27.5

40

EcoRI
Calculated
kilo base
pairs
19.1

42

23.1

44

16.9

21.2

50

14.0

12.3

56

9.4

60

10.3

7.4

65

8.8

7.2

65

6.6

70

7.6

5.8

67.5

8.2

6.8

80

4.4

72

7.1

5.6

70

7.6

5.6

111

2.3

75

6.5

4.9

120

2.0

90

4.1

3.5

Distance
(mm)

kilo base
pairs

Distance
(mm)

Actual
kilo base
pairs
24.8

BamHI
Calculated
Distance
kilo base
(mm)
pairs
45
16.3

16.8

Actual kilo
base pairs

F3. This table shows the distance (mm) from the top of the gel to each part of the banded pattern, the
estimated number of kilo base pairs, and the actual number of kilo base pairs. The distance was measured
from the ideal gel picture above (F1). The calculated kilo base pairs were determined by the linear
equation for the best fit line in the graph above (F2). The actual kilo base pairs were provided by the
teacher after the lab was completed.

Discussion
The results of this lab proved that my hypothesis was correct: each restriction enzyme did
create a different DNA fingerprint for lambda DNA. Restriction enzymes do cut DNA into a
variety of sizes because the certain sequence that the enzyme is looking for could appear any
number of times, and it does not have to be perfectly spaced out. The restriction enzymes will cut
DNA in different places regardless of how many times the required sequence appears.
The shorter DNA fragments did move farther through the gel than bigger pieces. The
amount that the fragments move is based on length. The smaller DNA fragments are easier to
move through the gel than longer pieces. The spaces between the bands in the DNA fingerprint
prove that the DNA was cut into varying sizes because some move more than others.

There were many sources of error that were present when we did this lab. The linear
equation from the logarithmic graph (F2) was not completely accurate because the best fit line
did not touch every single point on the scatter plot. Our teacher gave us the actual base pair
values so we could see the difference. Another error was that we did not have access to a
micropipette and we had to use less accurate pipettes. They were not very effective and caused
some of the lambda DNA and restriction enzyme mixtures to miss the wells in the gel and get
mixed into the buffer.
Another big error was that the gels were supposed to be run through the electrical current
for about an hour and a half. We ran out of time and only ran the gels for twenty minutes. We
also stained them for too long after we took them out of the electrophoresis box. We had no UV
light, which was needed to properly see the banded pattern for the type of dye we used. All of
these factors contributed to why no DNA fingerprint was present on our gel.
References
1: Biggs, Alton, Whitney Crispin Hagins, William G. Holliday, Chris L. Kapicka, Linda
Lundgren,
Ann Haley MacKenzie, William D. Rogers, Marion B. Sewer, Dinah Zike. Glencoe
Science Biology. Columbus: McGraw-Hill Companies, Inc., 2012. Print.
2: Freyer, Greg A. and David A. Mickles. DNA Restriction Analysis Kit. Burlington: Carolina
Biological Supply Company, 1995. Print.
3: Hendrickson, Kirstin. The Uses of Restriction Enzymes. Livestrong.com. Demand Media,
Inc., 1 July 2015. Web. 18 May 2016.
4: Restriction Enzymes. ASU School of Life Sciences. Arizona Board of Regents, 2014. Web.
18 May 2016.

5: Restriction Enzymes. Biotech Learning Hub. The University of Waikato, 20 November

2007. Web. 18 May 2016.
6: Restriction Fragment Length Polymorphism. NCBI. NCBI, 2014. Web. 19 May 2016.