Restriction Endonuclease Digestion of

Plasmid DNA
J.P. Latonio, R.E. Loquellano, N. Lustre, M.M. Manalo, E. Marasigan, and F.
Marzan
4Bio5, College of Science, University of Santo Tomas, Espaa, Manila

Summary
Keywords:
Restriction endonuclease,
pBR322, ethidium
bromide, agarose gel
electrophoresis

Restriction endonucleases are enzymes that cleave the sugar-phosphate

backbone of DNA, they are mostly isolated in bacterias, wherein they
also act as defense mechanism for the organism. For this experiment,
the restriction enzymes used are BamHI, EcoRI, HindIII, PstI, ScaI,
SalI, SapI, StyI. In the restriction digestion of the plasmid, five
combinations of restriction enzymes were used and incubated in a dry
block heater before adding gel-loading dye and loaded separately in 1%
agarose gel. To visualize the sizes of the fragments cleaved during the
plasmid digestion, samples were electrophoresed with ethidium
bromide as their staining medium. Of the eight wells loaded with
samples for experimental values, only lanes 6, 7, and 8 showed bands
under UV light. These lanes only exhibited a single band, which
represents the cleaved fragment from one of the enzymes at the same
time indicating an error of the differences between the displayed bands
with the number of expected bands. Lanes 1-5 did not display any
bands at all which may be due to DNA degradation or DNA
denaturation from nuclease contamination and excessive heat exposure,
respectively. The fragment sizes of the cleaved segments were not
determined as well due to the DNA ladder bands lacking inaccuracy.

Introduction
Restriction
enzymes
are DNA-cutting
enzymes found in bacteria. Because they cut
within the molecule, they are often
called restriction endonucleases. A restriction
enzyme recognizes and cuts DNA only at a
particular sequence of nucleotides called its
recognition site. The rarer the site it recognizes,
the smaller the number of pieces produced by a
given restriction endonuclease. Some of the

most common restricition endonucleases are

BamHI from Bacillus amyloliquefaciens, EcoRI
from Escherichia coli and Sall from
Streptomyces albus. Each of these restriction
endonucleases have their own unique
recognition
sites
but
enzymes
called
isoschizomers can have the same recognition
site.
pBR322 is a plasmid DNA isolated from E. coli.
This molecule is a double stranded circle having
4,361 base pairs. The plasmid pBR322 was one

of the first EK2 multipurpose cloning vectors to

be designed and constructed (1976) for the
efficient cloning and selection of recombinant
DNA molecules in Escherichia coli (Balbas, et
al., 1986).
Agarose gel electrophoresis is the standard lab
procedure for separating DNA by size (e.g.
length in base pairs) for visualization and
purification. Electrophoresis uses an electrical
field to move the negatively charged DNA
toward a positive electrode through an agarose
gel matrix. The gel matrix allows shorter DNA
fragments to migrate more quickly than larger
ones. Thus, the length of a DNA segment can be
accurately determined by running it on an
agarose gel alongside a DNA ladder which is a
collection of DNA fragments of known lengths.

the wells from left to right starting with the

DNA ladder on lane 1 and the sample without
restriction endonuclease on the extreme right.
The electrophoresis apparatus was covered with
the anode on the side of the wells. The
electrodes were attached to the power supply set
at 100V 250mA 50W. When the gel loading dye
moved up to one half, the power supply was
turned off. The gel was removed gently from the
electrophoresis apparatus and was transferred to
the developing tray containing 10uL ethidium
bromide per 100mL buffer. The gel was then
transferred to the gel documentation system. R f
values were measured for all the samples and the
digital images of the gel were recorded. Lastly,
the gel was immersed in hypochlorite solution
overnight before it was discarded.
Results and discussion
Ladder 1

8 control

Materials and Methods

Restriction Digestion
Distilled water, 10x buffer, restriction
endonuclease and pBR322 DNA were added to
each of the five 1.5mL microcentrifuge tubes. To
a separate microcentrifuge tube, all the reagents
were added except for the restriction
endonuclease. Different combinations of
restriction endonuclease were assigned per
group. The mixture was then incubated for one
hour at 37C in a dry block heater. After
incubation, 2 uL gel loading dye was added to
each mixture and was loaded separately in 1%
agarose gel. 50bp DNA ladder was placed in
lane 1 and electrophoresis was performed at
100V 250mA 50W.
Agarose Gel Electrophoresis
One gram agarose powder was dissolved in 100
mL 1x TAE buffer in a microwave oven. The gel
was cooled to approximately 60C and was
poured in the gel casting tray. The comb was
placed in position and the gel was solidified. The
comb was then removed gently. The gel casting
tray was placed into the submarine gel
electrophoresis system. The solidified gel was
covered with TAE buffer up to the maximum
mark, which is a few mm from the upper surface
of the gel. The samples were then loaded into

Figure1
The results of the gel are shown in Figure 1.
The number of restriction enzymes added to the
microcentrifuge tubes corresponds to the
number of expected bands to be seen, which
then signifies the number of segments formed
after the cleavage. In the figure shown, only
lanes 6, 7 and 8 showed bands, which confirms
that different enzymes cleaved the plasmid,
although the expected results were not achieved.
Ideally, lane 6 should have two bands
representing the two segments cleaved by the
two enzymes namely Bam HI and Eco RI as
well as lane 7 which should have two bands

showing fragments cleaved by Sca I and Sal I

and lane 8 should have three bands because of
the three enzymes: Pst I, Bam HI and Sca I.
The first well is reserved for the DNA ladder
that serves as a visual reference for the
determination of fragment sizes formed by the
restriction enzymes. Each band represents a
number corresponding to the length of segments
according to base pairs. In the results shown still
in Figure 1, bands in the DNA ladder are
accumulated at one portion of the gel making it
difficult for the bands found on the succeeding
wells to be compared with for their sizes.
Bands from lanes from 1-5 are not observable on
the same figure. One known error that
contributes to this is the insufficiency of quantity
or concentration of DNA loaded on the gel.
Another sources of error could be nuclease
contamination of the microcentrifuge tubes
leading to DNA degradation and high heat
standards exposure that may cause DNA
denaturation.

Figure 2

The plasmid map of pBR322 in Figure 2

indicates the restriction sites (displayed in
number) of the different enzymes. This can be

used to measure the size of DNA fragments

cleaved by a particular restriction enzyme. There
are a total of 4,361 base pairs in pBR322 and
therefore all calculated segment lengths in base
pairs based on the combination of enzymes
should sum up to 4,361.
Acknowledgements
Acknowledgement: The group is thankful to Ms.
Asia Abdulmajid, Asst. Prof. Josefino Castillo &
Asst. Prof. Michael Bahrami-Hessari for guiding
us in doing the experiment.
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