Gel Electrophoresis

Nate Saar
Honors Biology
5/23/16
Period 5

Introduction

Restriction enzymes are enzymes that cut the DNA molecule in a specific area, the
enzyme searching for a specific code or sequence to cut (Biotechlearn.org). The restriction
enzymes are used for DNA cloning, gene technology, inserting genes into a genome, as well as
criminal and paternity cases.
Gel electrophoresis is method used to separate and analyze DNA and RNA. The DNA
fragments are separated and measured by length (Wikipedia.org). It is used for separating the
DNA according to size and when the test is done the scientists can match up the DNA with the
original strand. Gel electrophoresis works because DNA is negatively charged and they are
attracted to the positive electrode, which causes the DNA to move through the gel, although at
different rates depending on their size. RFLP gel electrophoresis is how the DNA is separated
and cut into different fragments based on restriction enzymes. It is used in criminal cases and
paternity cases in order to match the DNA fragments with the original sample
(ncbi.nlm.nih.gov).
The purpose of this lab is to see and determine if the different restriction enzymes cut
DNA into different sized fragments or the same sized fragments. We also wanted to get practice
with restriction enzymes and gel electrophoresis. Finally, we wanted to create a logarithmic
graph with known data to figure out the other lengths of our DNA fragments created by different
restriction enzymes cutting them.
The independent variable in the lab was the DNA with the restriction enzyme. The
dependent variable was the actual distance travelled. Finally, the control group was the DNA

without any restriction enzyme applied. The DNA will show different fragments and cuts,
depending on the different restriction enzymes used.
Materials
1) Agarose Gel
2) Buffer Solution
3) Lamba DNA
4) Restriction Enzymes (EcoRI, BamHI, HindIII)
5) Micropipettes
6) Micropipette tips
7) Hot Plate
8) Eppendorf Reaction Tubes
9) 50 mL Beakers
10) 1000 mL Flasks
11) Electrophoresis Chamber
12) Graduated Cylinder
13) Microcentrifuge
14) Vortex
15) Ethidium Bromide Stain
16) Loading Dye
17) Gloves
18) Goggles
19) Staining Trays
20) Ultraviolet Light Source

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 table below as a checklist while adding reagents to each restriction. 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.
Tube
B
E
H
-

DNA
4 uL
4 uL
4 uL
4uL

Buffer
5 uL
5 uL
5 uL
5 uL

BamHI
1 uL
-------------------------

EcoRI
-------1 uL
---------------

HindIII
----------------1 uL
---------

H2O
---------------------1 uL

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 minutes at 37 C.
5. Teacher premade agarose gel
6. 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 microcentrifuge.
7. Use micropipette to load contents of each reaction tube into a separate well in gel.
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
bubbles 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.
8. 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 the same channel of power supply.
9. 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 positive pole
of electrophoresis apparatus.
10. 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.
11. Allow the DNA to electrophorese until the bromophenol blue band nears the end of
the gel.
Turn off power supply, disconnect leads from the inputs, and remove top of
electrophoresis chamber.
12. 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 Sample

Ideal Restriction Digest of DNA

HindIII
Dis.
41 mm
45.5 mm
57 mm
67 mm
81 mm
113 mm
120.5
mm

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

EcoRI
Dis.
43 mm
46.5 mm
63 mm
71 mm
76 mm
91 mm

Cal. bp
19.046
17.055
10.134
7.874
6.725
4.190

Act. bp
24,756
21,226
7,421
5,804
5,648
4,878
3,550

BamHI
Dis.
47 mm
53 mm
65 mm
67 mm
73 mm

Cal. bp
16.788
13.893
9.515
8.933
7.393

Act. bp
16,841
12,275
7,233
6,527
5,508

The table above describes the movement of the DNA in the ideal gel sample, which was
given. For each restriction enzyme the distance of the movement was recorded as well as the
calculated base pair number and the actual base pair number.

Distance Traveled by Each Fragment According to Size

1.6
1.4
1.2

f(x) = - 0.01x + 1.87

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 Traveled by Fragment (mm)

The chart above shows the information that is given in the table. Each point is
demonstrated by the distance traveled by the fragment and the fragment size. The line of best fit
is also given, which is shown by the linear equation.

Discussion
The hypothesis involving the DNA restriction enzymes was correct. Each restriction
enzyme cut the DNA fragments differently and each fragment moved through the gel differently.
Students learned that the restriction enzymes do cut DNA at different locations because each
enzyme looks for a different code in the DNA, signaling them to cut in that area. The larger
fragments travel less distance in the gel because it is larger and harder to move, but the smaller
fragments move through the gel much easier allowing them to move farther in the gel. Some of
the sources of error were that the gel used was a picture, which is different than a regular gel and
most likely in the measuring of the DNA fragments on the ideal gel picture.

References

Biotechlearn.org
biotechlearn.org.nz/themes/dna_lab/restriction_enzymes

Wikipedia.org
https://en.m.wikipedia.org/wiki/Gel_electrophoresis

ncbi.nlm.nih.gov
www.ncbi.nlm.nih.gov/genome/probe/doc/TechRFLP.shtml