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Restriction Enzyme Mapping of

Plasmid DNA and Agarose Gel


Electrophoresis
By Connor Thornton
Keith Herrera, Kristina Wright, Dhruv Patel
Section 9

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Connor Thornton

IntroRestriction Enzyme Mapping- In 1979 Werner Arben was awarded the Nobel Prize in
Physiology or Medicine for his research in discovering restriction enzymes. He
worked to discover the method in which viral DNA from certain bacteriophages
performed poorly in some bacteria and stronger in others of the same species.
Using E. coli Arben and Linn identified in 1969 that the virus DNA prevented the
infected host from restriction and thus preventing the virus DNA from being broken
down. They proposed that the genome contained specific sites of restriction and
were able to identify one which they named EcoB. Hamilton Smith and Kent Wilcox
soon, in 1970, identified the restriction enzyme Hindll in Haemophilusiinfluenzae
which is able to break down foreign DNA but will leave the host DNA intact. Later
that year, Smith and Thomas Kelly identified the nucleotide sequence that Hindll
cleaves(Pray, 2008). By using certain restriction enzymes DNA can be cleaved at
particular points. Restriction enzymes may also cut DNA so that the 3 and 5 ends
are both identical and have no polarity. This allows the DNA to bind to plasmids in
either of two directions (Cannons et all, 2008).
Agarose gel electrophoresis- Agarose gel works by creating an charge separation on
each side of the gel. A negative front is created on one side near the wells and a
positive front on the other. The negatively charged DNA molecules will then move
against the magnetic field created and travel through the gel towards the positive
front. The gel is a porous mixture that hinders the movement of the DNA such that
the smaller DNA fragments would travel further than long fragments. When the
charged fronts are removed, thus removing the magnetic field, the DNA molecules
are stay put and cease movement. This process allows the DNA to be sorted by
weight. Agarose gel was used rather than agar, polyacrylamide or composite
agarose-acrylamide gels as agarose gel causes less deviations between linear and
curved DNA (Stellwagen, 2009).
Ethidium Bromide (EtBr) is used to aid in the visualization of DNA post
electrophoresis through agarose gel. EtBr is visible using UV light and will bind to
DNA molecules allowing DNA bands to be clearly identified. The EtBr does increase
the weight the DNA slightly but not enough to prevent identification using standard
MW markers and the deviation is proportional to the size of the molecule causing
large molecules to have the largest deviation(Sigmon J, Larcom LL, 1996). In this
experiment this deviation will not hinder results as the DNA molecules used are not
large enough to show a notable irregularity. The hypothesis is that the fragments
provided by using the enzymes EcoRV and Bcll are able to identify the orientation of
the cDNA in the plasmid reliably.

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ResultsBand Identification- The two enzymes were gathered to cut the DNA at the Bcll and
the EcoRV respectively in order to identify the orientation of the cDNA insert as it
attached to the plasmid. The Agarose gel electrophoresis, figure 1, showed only four
bands for the MW marker in lane one which is likely due to the 564 and 125 bp
fragments running too far down the gel and becoming unidentifiable. The lack of
bands in the uncut plasmid in lane 2 shows that the plasmid did not travel through
the agarose gel likely because it is too large due to its ring structure. Only two
bands are visible in the plasmid 1 lanes but a third is likely hidden around the same
way the MW marker bands are missing since the missing band has an expected bp
of 302 which is around half way between the missing MW marker bands of 564 and
125.
Figure 1: Agarose Gel Image: A photo-image of the Agarose gel under UV light. The
migration of the bands was measured from the center of the wells to the end of the
image since the dye front was not visible.

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Table 1: Expected Digest Patterns: A chart of the predicted fragment sizes after
addition of enzyme. The plasmid contains 3919bp combined with the insert of
1622bp for a total of 5541bp.
cDNA Orientation
Enzyme
Expected # of
Fragments
Size of Fragment 1
(bp)
Size of Fragment 2
(bp)
Size of Fragment 3
(bp)

1
Bcll

2
Bcll

1
2
EcoRV EcoRV

302

302

916

779

1040

2281

4625

4762

4199

2958

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Connor Thornton
Table 2: Standard Data: Data collected from the known bp of the MW marker as
well as the distance traveled through the Agarose gel (figure 1). The final two bands
were not identifiable and thus not recorded.
Standard Marker Data
I
D # of BP Migration
1
23130 0.38/14
2
9416 3.75/14
3
6557 4.41/14
4
4361 5/14
5
2322 7.2/14
6
2027 7.65/14
7
564
8
125

Rf
0.03
0.27
0.32
0.36
0.51
0.55

Figure 2: Standard Graph: Graphic representation of Table 2 plotting Rf versus the


bp of the MW marker. The data follows an exponential curve and is shown with a
logarithmic scale.

Standard Marker Data


100000
10000
1000
DNA Size(bp)

f(x) = 28302.31 exp( -4.82 x )


R = 0.98

100
10
1
0.00

0.10

0.20

0.30

0.40

0.50

0.60

Relative Mobility(Rf)

Band Migration and Size- The bands in lane three of the gel, figure 1, are measured
to be around 4118 and 1571 bp in length (table 3) which is close to the predicted
4199 and 1040 bp (table 1) for plasmid 1 in orientation 1. The fourth lane showed
bands of 3315 and 2669 bp which are likely to be orientation two as the expected
fragment deviation was 677 bp and the distance between the two bands is around
646 bp. This is the shortest distance between any of the expected fragments in one
lane. The fifth lane had two bands of 4534 and 881 bp which compared to lane six
with two bands around 5240 and 1359 bp shows that lane five has a larger

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Connor Thornton
fragment and a smaller fragment than lane six which can identify them as
orientation two and one respectively.
Table 3: Plasmid Digestion Data: Distance traveled by each of the plasmid
fragments through the Agarose Gel shown in figure 1 with distances measured in
cm. The size of the fragments was calculated using figure 2 and the equation y =
28302e-4.819x.
Gel
lane

Plasmi
d#

RE

Size
(bp)

Rf

2
3
3
4
4
5
5
6
6

5.62/1
1 4
8.45/1
1 4
6.23/1
2 4
6.91/1
2 4
1 5.3/14
10.11/
1 14
4.92/1
2 4
8.86/1
2 4

0.40
0.60
0.45
0.49
0.38
0.72
0.35
0.63

4117.
86
1570.
72
3315.
07
2668.
78
4534.
49
880.9
6
5239.
8
1359.
29

cDNA Orientation- The lanes three to six were calculated using the agarose gel
image, figure 1, to suggest the orientation of lanes three and six, plasmid one, to be
in orientation one and two respectively while lanes four and six were calculated as
orientation one and two respectively. Each of the lanes of the same plasmid
orientation identify as separate orientations and an error may have occurred. Most
likely the closeness of the bands in lane five and six are due to minor errors
involving the agarose gel and the poor choice of using EcoRV as an enzyme as it
produces two fragments which are around 200bp different in each orientation of the
cDNA. The Bcll enzyme is much more reliable and is more trustworthy to point out
plasmid 1 as orientation one and plasmid two as orientation two. Further
experiments possibly involving different enzymes are needed to provide more
certain indication to the identification of the orientation of cDNA in each plasmid.

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Connor Thornton

References:
Pray, Leslie A. Restriction Enzymes. Nature Education. 2008.
<www.nature.com/scitable/topicpage/restriction-enzymes-545>
Sigmon J, Larcom LL. The effect of ethidium bromide on mobility of DNA fragments
in agarose gel electrophoresis. Electrophoresis. Oct 17, 1996.
<www.ncbi.nlm.nih.gov/pubmed/8957173>
Stellwagen, Nancy C.. Electrophoresis of DNA in agarose gels, polyacrylamide gels
and in free solution. Electrophoresis. June 30, 2009.
<www.ncbi.nlm.nih.gov/pmc/articles/PMC2757927>
Pollenz, Kimble, Cannons. Experiments in Cell Biology. 2008.

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