October 7th, 2017

Restriction Digestion Long Report

MacKenzie Olbrys

1. Introduction

Restriction digestion is a common technique used in biochemical and molecular and cellular
biological experiments. Restriction digestion requires restriction enzymes in order to cut DNA
into fragments. Restriction endonucleases are natural enzymes that exist inside the cell to protect
the cell from any invasion of possibly harmful foreign DNA.1 The homodimer enzymes function
by cleaving the double-stranded DNA at a short, usually palindromic, 4-8 bp sequences.2 When
cleavage occurs, a 5’ phosphate and a 3’ hydroxyl overhang is produced in either a blunt end or
sticky end cut. The purpose of these cuts in restriction digestion are to allow ligation to a desired
sequence and transformation of the product into other cells for further testing.3 In this study,
EcoRI and HindIII were used to cut the 2686 bp circular DNA plasmid pUC19. EcoRI recognizes
the sequence GAATTC on both strands of DNA and cuts between guanine and adenine located
at 396 bp on the pUC19 plasmid. HindIII recognizes the sequence AAGCTT on both strands of
DNA and cuts between the two adenines located at 447 bp on the pUC19 plasmid.4 In order for
the enzymes to be successful in cleavage, the correct pH, temperature, buffer, and metal ions
must be present. Any change in conditions of the environment surrounding the enzyme could
cause conformational changes in the protein which decreases the enzyme’s ability to attach to the
substrate of DNA and cleave. Both EcoRI and HindIII are type II restriction enzymes that require
divalent metal ions in order to not only bind to DNA, but also to cleave. Metal ions, like
magnesium used in this study, most likely serve a catalytic role in restriction enzymes, and
therefore, are vital to success for restriction digestion and must be included in the reaction
conditions.5 When two enzymes are used to cleave one plasmid, the conditions must be adjusted
to provide optimal efficiency of both enzymes. In NEBuffer 3.1 at 37 ℃, EcoRI and HindIII
have 50% activity.6 A double digest using EcoRI and HindIII should cause two nicks in the
circular plasmid to produce two linear bands of DNA at 51 bp and 2635 bp. Single digests should
cause one nick in the plasmid to produce a linear DNA of 2686 bp as controls.

2. Materials and Experimental Procedures

2.1 Restriction Digestion

The heat block was set to 37 ℃ and deionized water (ddH2O) was added to the wells. In four
different 500 µL centrifuge tubes, two µL of 10× restriction buffer 3.1 containing 100 mM NaCl,
50 mM Tris-HCl, 10 mM MgCl2, and 100 µg/mL BSA at pH 7.9, 11-13 µL ddH2O, and five µL
of pUC19 plasmid prepared by miniprep. Once the reagents above were added, the solution was
vortexed and centrifuged. One µL of both enzymes EcoRI and HindIII were added to tube one.
One µL of HindIII was added to tube two. One µL of EcoRI was added to tube three. No
restriction enzymes were added to tube four. Tubes two, three, and four served as controls for the
double digest. The total volume of all tubes equaled 20 µL. More or less ddH2O had to be added
depending on the enzyme amount to total 20 µL. The four tubes were placed in the heat block for
one hour.
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2.2 Gel Electrophoresis

A 2% agarose gel was made by adding 1.2 g of Calbiochem agarose to 60 mL of 1× TBE

buffer and heating in 10 second intervals to fully dissolve. Five microliters of ethidium
bromide was added to the cooled solution prior to pouring the gel. 1× TBE buffer was loaded
into the chambers of the gel electrophoresis apparatus and on top of the gel. Five microliter
samples of the four restriction digestion products mixed with one microliter of Promega 6×
loading dye were placed in four different wells of the gel. Five microliters of both Promega
100 bp DNA ladder and Fisher Bioreagents 1 kb DNA ladder were added to either side of the
gel. Ten microliters of the previous PCR samples were also loaded. The gel ran for 70 min at
90 V. The gel was visualized at UV 302 setting in the Axygen UV-Vis machinery as shown
in Figure 1.

3. Results

3.1 Restriction Digestion Analysis

The pUC19 plasmid double digestion and single digestion controls appear to have produced
DNA fragments about 2500-2600 bp in length as shown in lanes two, three, and four of Figure 1.
The control with no restriction enzymes added in lane five produced a DNA fragment that
appears to be about 1900 bp in length as compared to the Fisher Bioreagent 1 kb DNA ladder.
Linear regression analysis was performed on both the 100 bp DNA ladder and the 1 kb DNA
ladder by measuring the distance traveled from the center of the starting well to the center of the
DNA band as shown in Tables 1 and 2, respectively. From the distance traveled and log of
molecular weight, linear regression lines were composed as shown in Figures 2 and 3. The DNA
fragment in lane two was calculated to be 2450 bp. The DNA fragment in lane three was
calculated to be 2640 bp. The DNA fragment in lane four was calculated to be 2560 bp. The
undigested DNA fragment in lane five was calculated to be 1940 bp. All of the calculations for
the lanes 1-5 were performed using the linear regression line and equation shown in Figure 3.

4. Discussion

The DNA fragments expected to be seen in a double digestion with EcoRI and HindIII are 51
bp and 2635 bp both presented in the same lane. In the double digestion, there was only one
fragment observed at 2450 bp. In a single digestion using either EcoRI or HindIII, a linear
fragment of 2686 bp is expected. In the single digestion with HindIII, the DNA fragment was
2640 bp. In the single digestion with EcoRI, the DNA fragment was 2560 bp. Although the
number of base pairs calculated in relation to the 1 kb DNA ladder were not this size expected,
the relative movement of each band is as expected. The double digest products should move a
greater distance on the gel due to the smaller linear fragments produced by two cuts in the DNA.
The single digest products should be similar in size to each other due to one cut in the DNA
causing a linear form of the plasmid. The single digest products should be larger than the double
digestion products as supported by the gel image where the single digest products did not travel
as far. The difference in the actual sizes between expected and observed could be attributed to
the length of time the gel is ran. The longer the gel is run, the more separation that can be
observed in the DNA ladder. With better separation, the distance traveled measurement would be
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much more accurate. With just running the gel for 70 min, there seems to be possible faint bands
in the double digest lane which could be the smaller fragment that has not yet separated from the
larger fragment. With a longer run time, the bands in the double digest may become more
distinguishable from each other. The undigested, circular pUC19 plasmid is expected to be 2686
bp, but was observed at around 1940 bp. This is due to the circular structure of pUC19 that has
the ability to supercoil. Supercoiled DNA will be more compact, and therefore, be able to move
faster through the gel which was observed.
Overall, the bands did give the expected results of movement in relation to each other. The
circular plasmid should move the farthest, then the double digested DNA, and then the single
digested DNA. In order to achieve better results with mathematical calculations of band size, the
gel should be run longer and the DNA ladders should be better visualized by adding more dye.
Also, to see more separation in the double digest products, restriction enzymes should be chosen
to produce cuts farther apart from one another to yield larger, more easily visible DNA
fragments.

5. Tables and Figures

5.1 Figures

Figure 1: Restriction Digestion with EcoRI and HindIII and Control Rroducts on 2% Agarose Gel. After running gel
electrophoresis for 70 min, the pUC19 DNA was visualized using Axygen UV-Vis machinery at UV 302 setting and staining with
ethidium bromide prior to solidification. Lanes 1-10 consist of the following consecutively: 100 bp DNA ladder, tube one double
digestion with EcoRI and HindIII, tube two single digestion with HindIII, tube three single digestion with EcoRI, tube four with
no restriction enzyme, tube two PCR sample from week 4, tube three PCR sample, tube eight PCR sample, tube nine PCR sample,
and 1 kb DNA ladder.
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Restriction Digestion DNA Analysis with

100 bp Ladder
12

Distance Traveled (cm)

10
8
6
4
2
0
2 2.2 2.4 2.6 2.8 3 3.2 3.4
log(molecular weight) y = -7.6137x + 27.323
R² = 0.9985
Figure 3: Linear Regression Analysis from 100 bp Ladder on 2% Agarose Gel. The measurements of distance traveled were
done on a saved picture of the gel measuring from middle of the starting cell to the middle of the DNA band that traveled. Using
excel, a linear trend line was created with the equation and the R2 value to indicate the strong relationship between the data and
the linear regression line. The y values represent the distance the DNA fragment traveled in centimeters. The x values represent
the log of the molecular weight in base pairs.

Restriction Digestion DNA Analysis with 1 kb

DNA Ladder
10.0
Distance Traveled (cm)

8.0

6.0

4.0

2.0

0.0
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
log(molecular weight) y = -7.3177x + 27.182
R² = 0.9862

Figure 2: Linear Regression Analysis from 1 kb DNA Ladder on 2% Agarose Gel. The measurements of distance traveled were
done on a printed picture of the gel measuring with a ruler from the middle of the starting cell to the middle of the DNA band that
traveled. Using excel, a linear trend line was created with the equation and the R2 value to indicate the strong relationship
between the data and the linear regression line. The y values represent the distance the DNA fragment traveled in centimeters.
The x values represent the log of the molecular weight in base pairs.

5.2 Tables

Table 1: Analysis of 100 bp DNA Ladder Movement on 2% Agarose Gel

Molecular Weight (MW) (bp) log(MW) Distance Traveled (cm)

200 2.3 9.8
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300 2.5 8.6

400 2.6 7.5
500 2.7 6.8
600 2.8 6.1
700 2.8 5.6
800 2.9 5.2
900 3.0 4.8
1000 3.0 4.4
1500 3.2 3.3

Table 2: Analysis of 1 kb DNA Ladder and Restriction Digestion Movement on 2% Agarose Gel*

Molecular Weight (MW) (bp) log(MW) Distance Traveled (cm)

300 2.5 9.4
500 2.7 7.5
700 2.8 6.2
1000 3.0 4.9
1500 3.2 3.6
2000 3.3 2.9
2500 3.4 2.5
3000 3.5 2.2
Double Digest: 2450 3.4 2.4
Single Digest (HindIII): 2640 3.4 2.1
Single Digest (EcoRI): 2560 3.4 2.2
No Restriction Enzyme: 1940 3.3 3.1
*Bands above 3000 were not included because the bands were indistinguishable from each other.
6. References

1. CHM 6610 Experiment 5 Protocol and Lecture Notes, Fall 2017

2. Pingoud, Alfred, Jeltsch, Albert (2001) Structure and Function of Type II Restriction
Endonucleases, Nucleic Acids Research, 29, 3705-3727.
3. Kuspa, Adam, Loomis, William F. (1992) Tagging developmental genes in Dictyostelium
by restriction enzyme-mediated integration of plasmid DNA, Proc. Natl. Acad. Sci., 89,
8803-8807.
4. (2017) NEBcutter V2.0. New England Biolabs.
5. Vipond, Barry, Baldwin, Geoffery S., Halford, Stephen E. (1995) Divalent Metal Ions at
the Active Sites of the EcoRV and EcoRl Restriction Endonucleases, Biochemistry, 34,
697-704.
6. (2017) NEBuffer Activity/Performance Chart with Restriction Enzymes, New England
Biolabs.
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6. Supplemental Information

6.1 DNA Sequencing

The sequence analyzed corresponds to Homo sapiens cystathionine gamma-lyase protein as

determined by NCBI BLAST program.

7. Acknowledgements

Professor Dr. Christine Chow, teaching assistants Fidelis Ndombera and Rabiul Islam, and
my colleague Alexsandra Cvetkovska all assisted in the understanding and execution of this
experiment.