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DNA Restriction Enzymes Lab

Nick Milas
Honors Biology
May 25, 2015
Period 8

Introduction:

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This lab report discusses the roles of restriction enzymes and how we experimented with their
ability to cut strands of DNA. Restriction Enzymes are a particular set of enzymes that cut
specific sequences of DNA. They recognize a sequence of nucleotides in the DNA around four to
six base pairs long. A primary uses of restriction enzymes is in biotechnology in order to cut
DNA into smaller strands making it easier to study the differences in fragment length among
specific individuals. Biotechnology can be used to modify an organisms genome (ASU School
of Life Sciences). With this, new DNA may be inserted in the host genome by manipulating the
same genetic material of choice using cloning methods to produce a DNA sequence and inserting
the product in the host organism. Restriction enzymes can also be used with RFLP in paternity
and criminal cases. The RFLP method of paternity cases is when the DNA isolated from the
sample is cut into fragments with the help of enzymes. RFLP stands for Restriction fragment
length polypmorphisms and are the differences in lengths among individuals of DNA fragments
cut by enzymes. The DNA fragments are separated by size with an electrical current. DNA
probes then identify the separated DNA fragments. Gel electrophoresis is a method of separating
DNA, RNA, and proteins based on molecular size. Molecules that are able to be separated or
pushed by an electrical field with a gel containing small pores. The pores allow molecules to
travel in the gel that is inversely related to their lengths. Gel electrophoresis involves an
electrical field with positive and negative charge on each end. DNA and RNA are negatively
charged molecules that will be pulled towards the positive end of the gel. Since proteins are
positively charged, they must fix proteins with a detergent called sodium dodecyl sulfate. This
causes the proteins to unfold into a linear shape coated with a negative charge. This make them
able to migrate toward the positive end of the gel to be separated. Finally after the molecules
have been separated with the use of gel electrophoresis, band representing molecules can be

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detected with the different sizes. DNA fingerprinting uses gel electrophoresis to distinguish
between samples of genetic material. In forensics, some people may be eliminated if their DNA
pattern does match the DNA found at the crime case while others may become suspects if their
DNA matches the person who did the crime (Scientific American). The purpose of this lab to
see if different restriction enzymes chop DNA fragments or the same sized fragments. This will
allow us get practice with restriction enzymes and gel electrophoresis. We will create a
logarithmic graph with the known data to determine the other lengths of our DNA fragments
created with the different restriction enzymes cutting them. The dependent variable is the band of
the pattern of the DNA fingerprint. The Independent variable is the different restriction enzymes.
The control group is DNA and water. If we take lambda DNA and cut it with four restriction
enzymes then we will see different lengths of the fragments.
Materials:

Aragose Gel
TBE Buffer Solution
Lamboda DNA
Restriction Enzymes (EcoR1, BamHI, HindIII)
Micropipettes
Micropipette Tips
Hot plate
Eppindorf Reaction Tubes
50mL beakers
1000mL flask
Electrophoresis chamber
Graduated Cylinder
Micro centrifuge
Vortex
Ethidium Bromide Stain
Loading dye
Gloves
Goggles
Staining Trays

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Ultraviolet Light Source

Sharpie

Procedure
A: set up restriction digest
1. 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.
2. 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.
3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a short pulse in
microcentrifuge.
4. 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
1. 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 next step can set undisturbed.
2. Carefully pour enough agarose solution into casting tray to fill to 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 while gel is still liquid.
3. Gel will become cloudy as it solidifies. DO not move or jar casting tray while agarose is
solidifying.
4. When agarose has set, unseal ends of casting tray. Place tray on platform or fel box so that
comb is at negative.
5. Fill box with tris-borate-EDTA (TBE) buffer, to level that just covers entire surface of gel.
6. Gently remove comb, do not rip wells
7. Make certain that sample wells left by comb are completely submerged. If dimples are noticed
around the wells slowly add buffer until they disappear.
8. The gel is now ready to load with DNA. If you will be loading the gel during another period,
your teacher will instruct you to cover the electrophoresis tank to prevent drying of the 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 microcentrifuge.
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.

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a. Steady pipet over well using two hands.

b. Be careful to expel any air in micropipet 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 bromphenal blue; the slower-moving, aqua name is xylene cyanol. Bromophen
blue 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:

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This displays the ideal gel of a DNA fingerprint of the three enzymes and the control.

Distance Traveled by Fragments Cut with HindIII

1.6
1.4
1.2

f(x) = - 0.01x + 1.85

Fragment Ssize (log kbp) 0.8

0.6
0.4
0.2
0
30 40 50 60 70 80 90 100110120130

Distance Travelled by Fragment (mm)

The graph above displays the distance traveled of the fragment compared to the fragment size in
in kilo base pairs for the Hindll restriction enzyme. . The data points are connected with a bestfit line with an equation.

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Distance Travelled and KBP Length

HindIII
Dis.(mm) Act. bp

EcoRI
Dis.(mm)

Cal. bp

BamHI
Act. bp

Dis.
(mm)
51.0
56.0
69.0
72.0
77.0

Cal. Bp

Act. Bp

40.0
27,491
48.0
15,617 21,226
14,206 16,841
43.0
23,130
67.0
8,576
7,421
12,133 12,275
55.0
9,416
76.0
6,456
5,643
8,051
7,233
65.0
6,557
81.0
5,541
4,878
7,324
6,670
80.0
4,361
94.0
3,659
3,530
6,256
5,626
111.1
2,322
119.0
2,027
The Data Table shown above contains the results of all three restriction enzymes. For HindIII, it
has the distance and the actual base pair. For EcoRI and BamHi, it displays the distance, actual
base pairs, and the calculated base pairs.
Discussion
After finishing the experiment, my hypothesis that the restriction enzyme cut the DNA into
different sized fragments was correct. . The DNA fingerprint displayed the difference in sizes
between the actual base pair and the calculated base pair. The chart, displayed in the results,
proves that the shorter fragments travel father through the gel. This is probably because the
shorter fragments are able to fit through the pores of the gel easier than the large fragments.
There were still errors that we encountered during our lab that may have influenced the results.
One error was that the gel should have been heated for an hour and a half long, rather than the
twenty minutes we heated it for. Also, no ultraviolet light was used during the experiment.
Another possible error could have been from calculator mistakes or incorrect measuring.

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Works Cited
DNA Restriction Analysis. Carolina Biological Supply Company. Print. 18 May 2016.

Gel Electrophoresis. Scitable by Nature Education. Web. 19 May 2016.

Restriction Enzymes. ASU School of Life Sciences. Web. 19 May 2016.

What is gel electrophoresis, and why is it so important for DNA testing and criminal cases.
Scientific American. Web. 19 May 2016.