DNA Restriction

Analysis Lab

Rylee Kopchak
Biology Period 8
May 14, 2016

Introduction
In this experiment, the scientists used restriction enzymes to cut DNA from the
bacteriophage lambda into fragments, which were then separated using gel electrophoresis.
Restriction enzymes, also known as restriction endonucleases, cut a DNA molecule at a
particular sequence known as the recognition site. Restriction enzymes are extremely important
tools for analyzing DNA. According to Askabiologist.asu.edu, The enzyme scans a DNA
molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this
recognition sequence, it stops and cuts the strand. Restriction enzymes have a widespread use in
many molecular genetics techniques such as mapping DNA, verifying specific DNA fragments,
and the generation of recombinant DNA molecules. Recombinant DNA molecules are DNAs
that consist of genes from two different organisms. Restriction enzymes also play an important
role in identifying DNA strains of a particular species. Restriction Fragment length
polymorphism (RFLP) analysis is commonly used to determine a change in the genetic sequence
of DNA cut by a restriction enzyme. RFLP is used to identify individuals in criminal and
paternity cases, identify specific mutations, and trace inheritance patterns.
Gel electrophoresis is a technique used in the lab to separate charged molecules like
DNA, RNA, and proteins according to size. Gel electrophoresis works by sending an electric
current across the gel so that one end has a positive charge, while the other has a negative charge.
Molecules migrate towards the opposite charge: for example, a molecule with a negative charge
would be pulled toward the positive end of the gel. Smaller molecules pass through the gel at a
quicker rate, and therefore, travel further than larger molecules. Gel electrophoresis enables
scientists to distinguish DNA fragments of different lengths. After gel electrophoresis is
conducted, fluorescent or radioactive dyes are added, allowing scientists to study the DNA.
Agarose gels (a complex sugar material) are typically used and placed into an electrophoresis

tank, where the electric current is sent through. The purpose of this lab was to analyze whether
different restriction enzymes cut DNA into different sized fragments. In addition, the scientists
wanted to practice working with restriction enzymes and gel electrophoresis. After the
experiment was completed, the scientists created a logarithmic graph (displaying the known
data) in order to calculate the lengths of the remaining DNA fragments.
Experiment Details
Independent Variable: The Independent Variable was the restriction enzymes used to cut the
DNA.
Dependent Variable: The Dependent Variable in this experiment was the fragment size and
distance the DNA fragments travelled in the agarose gel.
Control Group: The control group in this experiment was the DNA that contained no restriction
enzyme.
Hypothesis: It is hypothesized that the restriction enzymes will cut the DNA into fragments of
various sizes, causing them to travel through the gel at different rates.
Materials

Agarose gel
TBE Buffer Solution
Lambda DNA
Restriction Enzymes (EcoR1,
BamHI, HindIII)
Micropipettes
Micropipette tips
Eppendorf reaction tubes
50 mL beakers
1000mL flask

Electrophoresis chamber
Graduated cylinder
Microcentrifuge
Vortex
Ethidium bromide stain
Loading dye
Gloves
Goggles
Staining trays
Ultraviolet light source
Sharpie


Procedure
1. Label four 1.5mL 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 reaction. Read down each
column, adding the same reagent to all appropriate tubes; use a fresh tip for each reagent.

Tu

be

DN

A
L
L
L
4
L

Buf

Ba
mHI

1
L
-

fer
5
L
L

Eco
RI
-

L
5

1
L

Hin
dIII
-

1
L
-

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 minutes at 37C.
5. Retrieve a pre-made agarose gel.
6. 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 the 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.
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.
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 same channel of power supply.

9. Turn power supply on and set voltage as directed by your instructor. Shortly after the
current is applies, 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 in xylene
cyanol. Bromophenol blue migrates through the gel at the 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.
12. Turn off power supply, disconnect leads from the inputs, and remove top of
electrophoresis chamber.
13. Carefully remove casting tray and slide gel into staining tray labeled with your group
name. Take gel to your instructor for staining.
Results

*This picture shows an ideal agarose gel after gel electrophoresis.

After staining the dye with Bromophenol blue, the DNA
fragments become visible under UV light, and the scientist are
able to see the distance each fragment travelled.

Distance Travelled by Fragments Cut with HindIII

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

1.2
1

Fragment Size (log kilobase pairs) 0.8

0.6
0.4
0.2
0
20

40

60

80 100 120 140

Distance Travelled by Fragment (mm)

*This graph shows the distance travelled by the DNA fragments cut with HindIII in
millimeters. In addition, it displays a line of best fit and the correlation between fragment
size and the distance it traveled through the gel.

HindIII
Di

s.
(m
m)

42

46.
5

60.
5

A
c
t.
b
p
2
7
,
4
9
1
2
3
,
1
3
0
9
,
4
1

Di
s.
(m
m)

44

47

66

EcoRI
C
a
l.
b
p
1
9
,
7
7
9
1
7
,
9
9
3
9
,
8
8

A
ct
.
b
p
2
4,
7
5
6

Di
s.
(m
m)

50

2
1,
2
2
6

55

7,
4
2
1

67

BamHI
C
a
l.
b
p
1
6
,
3
6
8
1
3
,
9
7
0
9
,
5
7

A
ct
.
b
p
1
6,
8
4
1

1
2,
2
7
5

7,
2
3
3

6
1
4
70
6
75
7
5,
70
8
6,
,
,
6
,
5
5
4
4
7
2
5
3
3
1
7
7
9
0
83.
4
80
6
4,
75
7
5,
9
,
,
8
,
5
3
3
7
4
0
6
5
8
3
5
1
3
9
11
2
93.
4
3,
5.5
,
4
,
5
3
1
3
2
6
0
2
3
12
2
3
,
0
2
7
*This chart displays the measured distances travelled by DNA cut with each restriction
enzyme: HindIII, EcoRI, and BamHI. It also shows the actual number of base pairs that
formed each fragment, along with the number the scientist calculated in the experiment.
Discussion

After completing the experiment, the scientists hypothesis was supported. The

restriction enzymes did cut the DNA into fragments of different lengths, causing them to travel
various distances. The restriction enzyme HindIII cut the DNA into the most pieces (seven
fragments), causing them to travel 123mm through the agarose gel. EcoRI cut the DNA strand
into six fragments. As a result, the DNA travelled 93.4mm through the gel. Finally, BamHI cut
the DNA into only five fragments, travelling 75mm through the gel. Restriction enzymes cut
DNA at different locations because each of them has a distinct sequence of nucleotides that tells
it where to cut the strand. The more times the restriction enzyme cuts the DNA, the farther it

will travel through the gel during gel electrophoresis. This is because smaller fragments move
through the gel easier and quicker than larger DNA fragments.

Some mistakes that may have been made during the experiment include

measurement errors, and mistakes made while staining the gel. Mistakes in measurement may
have occurred while measuring the amount of restriction enzymes and DNA to put in the reaction
tubes. In addition, slightly inaccurate measurements could have been made when the scientist
measured the distance each DNA fragment travelled through the gel. Because of this error, the
calculated base pairs varied marginally from the actual number of base pairs. Finally, after dying
the gel with Bromophenol blue, the DNA did not appear under the light. This may have been
because the dye was ineffective or the light source was not strong enough.
Resources

DNA Restriction Analysis Lab Manual

https://askabiologist.asu.edu/restriction-enzymes

http://www.yourgenome.org/facts/what-is-gel-electrophoresis