Gel Electrophoresis Lab Report

Kaitlyn Grubbs
Honors Biology
May 15, 2016
Period 5

A restriction enzyme is a chemical that cuts DNA into fragments. These restriction
enzymes cut in a palindromic sequence of the DNA, meaning that the DNA is able to be read the
same way whether being read forwards or backwards. Restriction enzymes are also used by
scientists in selective breeding, the Human Genome Product, and genetic engineering. The
Human Genome Product, which took place in 2003, mapped out all of the 30,000 genes on the
forty-six chromosomes that can be found in humans. It also allowed for scientists to insert a gene
into an organism, such as to fix a gene that was not functioning correctly. Genetic engineering,
first completed in the 1970s, is technology that manipulates DNA. This process included
recombinant DNA, DNA from two different organisms, such as bacterial plasmid which is a
small piece of DNA. Through restriction enzymes, RFLPs, which are the restriction length
polymorphisms (HindIII, EcoRI, BamHI), are used in criminal cases and paternity cases. Gel
electrophoresis is the separation of DNA that is pushed by an electrical field through a gel that
contains small pores. The process of gel electrophoresis is used when scientists need to sort DNA
strands according to length. According to futurescienceleaders.org, DNA is negatively charged
because of all the phosphate groups in the backbone of the DNA. Therefore, DNA is attracted to
the positive electrode. As the pieces of DNA move through the gel, they will feel resistance.
Larger pieces of DNA will move at a slower rate than smaller fragments of DNA. According to
Ncbi.nlm.nih.gov, Restriction Fragment Length Polymorphism (RFLP) is a difference in
homologous DNA sequences that can be detected by the presence of fragments of different
lengths after digestion of the DNA samples in question with specific restriction endonucleases.
This can be used to help in criminal and paternity cases. As the fragments of DNA travel through
the gel, those that match the DNA from the parent/crime scene, proves that it is the same DNA.
If there were two suspects for a crime, the DNA that matches the DNA found at the crime scene
will be the culprit because the DNA matches. As for paternity cases, if the DNA from the child
matches the parents DNA, then they are the child of that parent, but, if the DNA does not match,
the two are not related. A DNA Fingerprint is biometric identification obtained by examining a
person's unique sequence of DNA base pairs; often used for evidence in criminal law cases,
according to Thefreedictionary. In short, each person has their own DNA Fingerprint that is
unique to them and no other person has the same one.
The purpose of this lab is to cut DNA of the bacteriophage by three different restriction
enzymes (HindIII, EcoRI, BamHI) into different fragments. These fragments are separated using
gel electrophoresis. The purpose is also to see if different restriction enzymes cut DNA into
different sized fragments or the same sized fragments as they travel through the gel. Also, we
want to get practice with the restriction enzymes and use of gel electrophoresis. This enables us
to create a logarithmic graph with the known data to discover the other lengths of the DNA
fragments that were created by the different restriction enzymes when they were cut. In this lab,
the dependent variable is the distance traveled by each fragment because it relies on the length of
the fragments, which can be changed and is the independent variable. The control group of this
experiment is the negative control, which is incubated without the endonucleases (HindIII,
EcoRI, BamHI). I predict that the smaller fragments will travel through the gel faster than the
larger fragments because it will be easier for a small fragment to travel through the pores of the
gel.

Materials List:

Agarose Gel

Buffer solution

Lambda DNA

Restriction Enzyme (EcorR1, 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

Procedures

Procedure A: Set Up Restriction Digest

1. Label four 1.5-mL tubes, which will be used to 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 appropriate tubes;
use a fresh tip for each reagent.
Tube
B

DNA

Buffer

BamHI
1 L

EcoRI
-

HindIII
-

H 2O
-

4 L

5 L

4 L

5 L

1 L

4 L

5 L

1 L

4 L

5 L

1 L

3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a
short pulse I a micro centrifuge.
4. Incubate all reaction tubes for a minimum of 20 minutes at 37 oC.
Procedure B: Cast Agarose Gel
1. Obtain a gel from teacher.
2. When agarose as set, unseal ends of casting tray. Place tray on platform of
gel box, so that comb is at negative (black) end.
3. Fill box with tris-borate-EDTA (TBE) buffer, to level that just covers entire
surface of gel.
4. Gently remove comb, taking care not to rip walls.
5. Make certain that sample wells left by comb are completely submerged. If
dimples are noticed around wells, slowly add buffer until they disappear.
6. The gel is now ready to load with DNA.
Procedure C: Load Gel

1. Add 1 L loading dye to each reaction tube. Mix dye with digested DNA by
tapping tube on lab bench, or with a pulse in microcentrifuge.
2. Use micropipette to load contents of each reaction tube into a separate well
in gel, aligned as illustrated in Idea 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 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.
Procedure D: Electrophorese
1. Close top of electrophoresis chamber and connect electrical leads to an
approved power supple, anode to anode (red-red) and cathode to cathode
(black-black). Make sure both electrodes are connected to the same channel
of power supply
2. Turn power supple on and set voltage as directed by instructor. Shortly after
current is applied, loading dye can be seen moving through gel toward
positive pole of electrophoresis apparatus.
3. The loading dye will eventually resolve into two bands of color. The fastermoving, 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.
4. 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 5 and 6.
5. Turn off power supple, disconnect leads from the inputs, and remove 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
Ideal Gel

This is the picture of the ideal gel used for measurements to make the charts located
below. The ideal gel was used rather than the constructed gel in the lab, which did not work and
the DNA was unable to be seen.
Chart of Distance Travelled by Fragments Cut wit HindIII

Distance Travelled by Fragments Cut with HindIII

1.6
1.4
1.2

f(x) = - 0.01x + 1.9

Fragment 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 (mm)

The graph above depicts the distance traveled in millimeters by the fragments cut with
HindIII, a restriction enzyme. Each fragment was measured in log kbp and is on the y-axis. The
distance traveled is measured and located on the x-axis. There is a line of best fit which travels
through the graph and a linear equation that was used in calculating the base pairs in EcoR1 and
BamHI.
Distances Traveled and Base Pairs
HindIII
EcoRI
Distance Actual bp Distance
Cal. bp Actual bp Distance
42mm
*27,491
43mm
20.413
*24,756
48mm
46.5mm
*23,130
47.5mm
17.711
*21,226
50mm
60.5mm
9,416
66mm
9.881
7,421
55mm
70mm
6,557
74.5mm
7.557
5,643
68mm
83.9mm
4,361
80mm
6.353
4,878
76mm
115.5mm
2,322
94mm
4.085
3,530
123mm
2,027
**564
**125
*Pair appears as single band. **Does not appear on this gel.

BamHI
Cal. bp
17.434
16.368
13.98
9.277
7.208

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

Located above is a chart that depicts the distances traveled by DNA fragments after being
cut by the various restriction enzymes. It includes the length of each DNA strand the calculated
and actual. The calculated base pairs were found using the linear equation y = -0.0137x + 1.899
and then solving. The solved number was then used in the inverse log, resulting in the calculated
base pair.

Discussion
1.

Analyze your results.

1.

Was your hypothesis correct?

2.

Do restriction enzymes cut DNA at different locations?

3.

What sized fragments travel farther through the gel?

4.

Sources of error

References
1.

List of at least 3 sources used in introduction.

2.

Cite lab manual for procedures.

3.

In-text citations are included.

Future Science Leaders: http://futurescienceleaders.org/researchers2012/2013/03/how-does-gelelectrophoresis-work/

Restriction Fragment Length Polymorphism (RFLP):
http://www.ncbi.nlm.nih.gov/genome/ probe/doc/TechRFLP.shtml