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DNA Module Lab


Siobhan Estabrook, Daniela Dinkins, Dr. Annette McGehee
BIO L111
Department of Biology
Suffolk University
Boston, MA 02114
April 29, 2015
Introduction
The purpose of this module is to isolate our plasmid from the bacteria and to determine if
it contains the gene of interest or if it does not. DNA contains genes that code for proteins.
Scientists can study genes and proteins by isolating the DNA of interest using DNA recombinant
technology or DNA cloning. DNA cloning isolates genes from one organism then inserts them
into a plasmid vector and then finally transfers the new plasmid back into bacteria. In this
experiment we are interested in using the process of DNA cloning to insert a gene of interest into
our bacterial plasmid. This will allow us to study the gene of interest. DNA cloning relies on
enzymes called Restriction Endonucleases, which identify specific DNA sequences and cut the
DNA to produce fragments of exact sizes (Thill).
In order to be able to reach the ultimate goal of isolating our plasmid and determining if it
contains the gene of interest or not, multiple experiments were necessary. To be able to determine
which restriction digests would allow us to differentiate between the sequences of plasmids that
contains the gene of interest and one that does not, we will work with the program ApE on the
computer. Using this program we will find restriction sites and calculate the fragment sizes.
Based off of this information we will choose a restriction digest we feel will work the best in the
experiment. For this experiment we chose Pvu II as our restriction enzyme.
After being given a pellet of E. coli bacteria that contains the two plasmids we will
isolate the plasmid DNA. Purification of the bacteria will leave us with just the plasmid DNA to
cut with the restriction enzyme we choose. We will perform a restriction enzyme digest to cut our
purified plasmid with our restriction enzyme. This will characterize our plasmids to ensure the
DNA was not mutated during the cloning process (Thill). To be able to separate out the DNA
fragments by their sizes we will use an agarose gel electrophoresis. This will also allow us to

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determine the sizes of the fragments and we will analyze this information to determine whether
our plasmid contains the gene of interest. To determine the size of the DNA fragments, we will
graph a calibration curve based on the log size and migration distance through the gel.
Experimental Procedure
Bioinformatics
Using the computer program ApE we gathered information using four restriction
enzymes. We first opened the DNA file named plasmid containing the sequence for the
plasmid without the gene of interest. Next to indicate that our DNA sequence is a circular DNA
fragment we clicked the button that read circular. After, under the Enzymes Menu we
clicked on Enzyme Selector. Next, we chose one of the four enzymes by highlighting its name
on the list and clicked on the Digest button. We repeated this for each of the four enzymes for
the files called plasmid and plasmid with gene of interest. Based off of this information we
were able to choose a restriction enzyme that would differentiate between two plasmids.
DNA Miniprep
To purify the DNA we first, using the E. coli sample of strain two we added of 250 ul of
the P1 solution to the sample. Next, we added 250 ul of the P2 buffer mixed this very well by
inverting the tube 4 to 6 times. We then added 350 ul of the N3 buffer and mixed this
immediately. Next we centrifuged this for ten minutes at 13,00 rpm in the micro centrifuge.
Using the supernatant we applied it to the QIAprep spin column by pipetting. We centrifuged this
again for sixty seconds. After we discarded the liquid that did not bind to the column, which was
in the collection tube. The DNA is left on the column. We then washed the QIAprep spin column
with 0.75 ml of the Buffer PE and centrifuged this for sixty seconds. Again we discarded the
flow through and centrifuged at full speed of an extra minute to remove residual wash buffer.
Next we placed the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute the DNA we
then added 50 ul (was supposed to be 30 ul) of Buffer EB or water to the center of each QIAprep
spin column. We let this stand for one minute and then centrifuged for one minute. Using the
NanoDrop mini spectrophotometer at 260 nm we determined the concentration and purity of our
sample. At the ratio of A260/A280 of 1.82 our concentration resulted in 277.3 ng/ul.
Restriction Enzyme Digest

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In order to cut the purified plasmid with our restriction enzyme Pvu II we completed a
restriction enzyme digest. First, we calculated how much our sample of DNA we would be using.
After calculations 1.1 ul was the total amount of DNA we would use. Next, in a new tube we
added 1 ul of the Cut Smart Buffer. We then added the 1.1 ul of DNA. Next, we added 6.9 ul of
water and then 1 ul of the Pvu II enzyme. This made the total volume 10 ul. We then centrifuged
this for one minute. Finally we incubated this at 37 degrees Celsius for one to three hours (Thill).
Agarose gel electrophoresis
After incubation we prepared the sample for the electrophoresis to separate the DNA by
size. we first prepared a sample without the restriction enzyme. We did this by adding the 1.1 ul
of DNA, 8.9 ul of water and 2 ul of the loading buffer to the uncut sample. We then centrifuged
the sample for one minute. Next, we worked with the sample of digested DNA by adding 2 ul of
the loading buffer and then centrifuged for one minute. Then we loaded both of the samples onto
the gel into our assigned lanes. The gel ran for fourty five minutes at 80 volts. After the gel run
was complete, the gel was stained with a dilute solution of ethidium bromide (Thill).
Data Analysis
Using the image of our gel, we used the information from that to determine the log size of
the DNA fragments in our sample. We used the 1kb ladder marker, which allowed us to compare
the log size of each band and the migration distance in millimeters. With this information we
created a graph and calibration curve with the migration distance plotted on the x-axis and the
log size plotted on the y-axis. We then used the equation from that graph to find the size of the
restriction enzymes by plugging in the migration distance for x. Now using this information we
assessed whether or not our plasmid contained the gene of interest (Thill).
Results and Discussion
In order to understand how restriction enzymes work, we first used bioinformatics with
the program ApE. We were able to electronically cut the DNA with four restriction enzymes
each. The electronic digest generated the sizes of the fragments (bps). Seen in Figure 1 is the
chart we filled out according to how each restriction enzyme cut the plasmids. From this
information we were able to decide which one we would want to use for our experiment. Based
on the chart we chose the Pvu II enzyme because it seemed to cut the DNA the most and would
generate a different number of DNA fragments. Pvu II had two recognition sites in the plasmid
without the gene of interest and three recognition sites in the plasmid with the gene of interest.

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Figure 1
This is the chart we completed according to how each restriction enzyme cut the plasmids. From
this information we were able to decide which one we would use for our experiment. Based on
the chart we chose the Pvu II enzyme because it seemed to cut the DNA the most and would
generate a different number of DNA fragments. Pvu II had two recognition sites in the plasmid
without the gene of interest and three recognition sites in the plasmid with the gene of interest.
Plasmid

Plasmid with

Restriction

# of recognition

Sizes of

gene of interest
# of recognition

Sizes of the

enzyme

sites in the

fragments by

sites in the

fragments by

Bg III
EcoRI
NotI

plasmid
0
1
1

digest(bps)
2961
2961
2961

plasmid
0
1
2

digest (bps)
3678
3678
2961

PvuII

2513

717
2513

448

792
373

After now knowing which enzyme we were going to cut with, we next moved onto the
DNA miniprep. We were given a pellet of E. coli bacteria from strain two. This contained one of
the two plasmids whose sequences we analyzed in the bioinformatics section. We then isolated
the plasma DNA from the bacteria. Before purification, our sample of E.coli contained proteins,
chromosomal DNA, RNA, and cell membranes. Our goal was to get rid of as many contaminants
as possible so the isolated plasma DNA would be free of these cellular components (Thill). The
first solution we used, P1 solution contained buffer and ribonuclease, which allowed for high
purity of the purified plasmid. The buffer P2 contained SDS that denatured cellular proteins and
membranes. It also contained Sodium Hydroxide that hydrolyzed the RNA and denatured the
chromosomal and plasmid DNA. The Buffer N3 neutralized the solution causing the plasmid
DNA to regain its original structure, but the chromosomal DNA did not. After centrifugation, the
supernatant contained the partially purified plasmid DNA. Using this supernatant we pipetted it
onto the QIAprep. The DNA was bounded to the column after centrifugation. After washing and

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centrifugation of the QIAprep containing the DNA, to ensure purification, we used the
NanoDrop next. The NanoDrop determined the concentration of our sample of DNA using the
extinction coefficient for double stranded DNA, relating absorbance at 260nm to DNA
concentration (Thill). Our final concentration was 277.3 ng/ul.
Now that the plasmid DNA is isolated we performed the restriction digest. Restriction
enzyme digests are used to characterize newly made plasmids to ensure that the DNA was not
mutated during the cloning process. (Thill). The restriction digest cut our purified plasmid with
Pvu II. In order to prepare the digest we had to calculate how much of our DNA we would use.
We divided 300 ng by our concentration of DNA 277.3 ng/ul to get 1.1 ul of DNA to be used in
the digest. The digest must have a volume that is less than 10% of the buffer. Seen in Figure 2 is
a chart of the components of our digest. 1.1 ul of the 277.3 ng/ul of DNA is used, 1 ul of the Cut
Smart Buffer, which is most effective with our enzyme Pvu II, 6.9 ul of water, and 1 ul of Pvu II
enzyme. All components were then centrifuged and incubated.
Figure 2
This is a chart of the components used for our digest. 1.1 ul of the 277.3 ng/ul of DNA is used, 1
ul of the Cut Smart Buffer which is most effective with our enzyme Pvu II, 6.9 ul of water, and 1
ul of Pvu II enzyme. The total volume is equal to 10 ul. All components were then centrifuged
and incubated.
Component
DNA 277.3 ng/ul
Cut Smart Buffer
Water
Pvu II (enzyme)

1.1 ul
1 ul
6.9 ul
1 ul

The next step was gel electrophoresis. Using the agarose gel electrophoresis we were able
to compare the sizes of our uncut sample and digested sample of DNA and we were able to
determine whether it contained the plasmid with the gene of interest or not. When the gel is
exposed to electricity this moves the negatively charged DNA downward towards the positive
pole. DNA has a negative charge from its negatively charged phosphate backbone this is why it
travels towards the positivity. As the DNA fragments move through the gel towards the positive
pole, the smaller fragments move faster than the larger ones, allowing the separation based on
their log size (Thill). After the DNA moved through the gel, the gel was removed from the
electric current and stained with a dye that binds to DNA. The staining allowed us to see the

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DNA. An image of our gel is seen in Figure 3. In figure 3 our samples were loaded into lanes 10
and 11. In lane 10 you can see the DNA sample that is uncut, and the digested sample in lane 11.
Here, you can see the uncut sample was cut twice, and the digested sample only once. According
to our prediction (Figure 1) Pvu II should have cut three times. Our data was inconclusive, but
based on other classmates data that used Pvu II as well, our plasma should have been without
the gene of interest. Therefore, our experimental results are not consistent with the predictions
we made based on the bioinformatics analysis (Figure 1). There are a few possible sources of
error. We could have accidently cut with Not I, but I do not think this is the most likely thing that
could have happen since we were very careful. Another more likely reason could be that the
DNA band is actually there in lane 11, but we did not load enough DNA onto the gel for it to be
visible.
After coming to these conclusions we used the information from the image of the gel to
determine the actual size of the restriction fragments. We measured the migration distance of our
bands by using the markers in millimeters, seen in the chart in Figure 4. Using the data from the
molecular weight markers chart (Figure 4) we generated a calibration curve (Figure 5). Here, we
plotted the migration distance on the x-axis and the log size (in base pairs) on the y-axis. Any
fragment size in the linear range can be estimated accurately. Seen in Figure 6 we used the
equation the graph generated: y=-0.0189x+4.447. We then plugged in the migration distance of
our restriction digest sample for x. This gave us y=3.54 which is the log size of our restriction
digest sample. Next we took the inverse log of 3.54 we got 3467 kbs, which is the size of our
restriction digest sample.
Figure 3
Here is an image of Gel 2 with our samples being loaded into lanes 10 and 11. In lane 10 you can
see the DNA sample that is uncut, and the digested sample in lane 11. Seen here you can see the
uncut sample was cut twice, and the digested sample only once. According to our prediction
(Figure 1) Pvu II should have cut three times. Our data was inconclusive, but based on other
classmates data that used Pvu II as well, our plasma should have been without the gene of
interest.

1 2 3

4 5

7 8 9 1 11
0

Figure 4
In this chart we measured the migration distance (mm) of each of the markers also seen in
Figure 1 Lane 1. Using this chart we created the calibration curve to determine the linear range to
then determine the size of our digested sample to see how far it traveled through the gel.
Size (kb)
10
8
6
5
4
3
2
1.5
1
.5

Migration Distance (mm)


27
32
36
38
43
48
58
65
78
95

Log size (bp)


4.00
3.90
3.78
3.70
3.60
3.48
3.30
3.18
3
2.70

Figure 5
The curve this graph creates gave us the equation necessary to determine the size of our
digested sample. Here, we plotted the migration distance on the x -axis and the log size (in base
pairs) on the y -axis. Any fragment size in the linear range can be estimated accurately.

Migration Distance vs Log Size from Gel

f(x) = - 0.02x + 4.45


R = 0.99
Log Size (bp)
Linear (Log Size (bp))
Linear (Log Size (bp))

Figure 6

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This chart shows the one band that was visible in our restriction digest. Using the equation from
the graph in Figure 5 y=-0.0189x+4.447. We then plugged in the migration distance of our
restriction digest sample for x. This gave us y=3.54 which is the log size of our restriction digest
sample. Next we took the inverse log of 3.54 we got 3467 kbs, which is the size of our restriction
digest sample.
Size (kb)
3467
References

Migration Distance

Log Size (bp)

(mm)
48

3.54

Thill, G., and McGehee A. Bio 111 Lab Manual, Suffolk University, Boston, MA 2015.