BTEC 1015 Plasmid Identification Report

Introduction
Plasmids are DNA molecules which are
often found in bacteria, but have been also
naturally found in archaea and eukaryotic
organisms. These circular DNA molecules
are separate from the main hosts' DNA and
hold genetic information for the production
of other proteins. The extra genetic material
may prove to be beneficial to the host ability
of survival or its direct demise.
Today, plasmids are used as vectors to
perform gene transfer and since DNA is
life's universal script it is possible to
transfers DNA sequences between different
organisms.
Proteins which cuts DNA strands at a
specific base pair location are denominated
Restriction Enzymes. As their name implies,
Restriction enzymes cleave the DNA
sequence it fits with making a cut in the
sugar phosphate backbone and one in the
nucleotide base pair. These enzymes are
used to "digest" plasmids or other DNA
sequences in order to cut at specific sites
which makes them have specific fragment
sizes that can later be recognized and further
studied.
Gel electrophoresis is the process in which
the genetic material (DNA or RNA) is cut
up in fragments with restriction enzymes,
these fragments will be set in an agarose gel,
separated by their size. With those results it
is possible to analyze the results and develop
a conclusion as to know what kind of DNA
(or RNA) sequence it is, based on the gel
electrophoresis results.
The aim of this laboratory experiment is to
use the process of gel electrophoresis with a
random unknown plasmid, already
"digested" by restriction enzymes, to obtain
data Based on that data it is possible to
compare and contrast the experiment's
results with the predictions of each plasmid
possible for being the unknown plasmid.
After fully analyzing results with
predictions, it should be possible to
determine which plasmid it is.
Gel electrophoresis is one of the many
laboratory process done in the field of
biotechnology used to recognize gene
sequences. The ability to learn how to run
complex processes such as these is a
requirement in order to understand further
biotechnology uses and applications.
In order to recognize the unknown plasmid
given to me. I digested the plasmids with
specific restriction enzymes and ran gel
electrophoresis, after which were analyzed
to determine what plasmid it was.

Methods
The code name of my plasmid was:
5212B58. The procedures are divided into
three parts: Restriction digest, Agarose Gel,
and Sample preparation for gel.

Restriction Digest
The assembling of restriction digest is the
lengthier part of the gel electrophoresis for it
must be carefully assembled and it must rest
for a minimum of one hour.
We begin by labeling five microfuge tubes
and label them: Control, HindIII, BglI,
EcoRI, and EcoRI+HindIII. After properly
labeling fill the calculated amounts of
substances as labeled in Chart 1.1
HindIII, BglI, and EcoRI required different
buffers in order to obtain maximum
efficiency. According with New England
BioLabs' Double Digest Finder webpage, it
helped to find the NEBuffer necessary to
have maximum efficiency for each
individual Restriction enzyme. Lack to
apply the proper buffer it would've resulted
in no full digest and may have required more
time. The program suggested to use 2.1
NEBuffer for HindIII and EcoRI restriction
enzymes. While BglI used 3.1 NEBuffer.
(NEBuffer, HindIII, BglI, and EcorRI were
obtained from New England BioLabs, all
rights reserved).
Tube Miniprep 10x
Buffer
dH2O BglI HindIII EcoRI Total
Volume
Control 3.3uL 2uL
14.7uL
----- --------- --------

20uL
HindIII 3.3uL 2uL
13.7uL
----- 1uL --------
BglI 3.3uL 2uL
13.7uL
1uL --------- --------
EcoRI 3.3uL 2uL
13.7uL
----- --------- 1uL
EcoRI+HindIII 3.3uL 2uL
12.7uL
----- 1uL 1uL
Chart 1.1: This chart labels the exact measurements used in the laboratory to assemble the
restriction digest.
After assembling the proper substances
labeled in Chart 1.1. The test tubes were
applied gently vortexed in order to mix the
substances and to produce a good digest.
The tubes will now go for a 37 degree
Celsius water bath incubation for 1 hour.
This is in order for the restriction enzymes
to obtain a stable environment in which they
can catalyze quickly.

Agarose Gel
While letting the microfuge tubes incubate
for one hour. We made a 50 ml agarose gel.
Since the ladder is a 1kbp (kilo base pairs)
ladder, the concentration of the gel must of
.8 concentration since that concentration will
allow for a range of 10000 base pairs (bps)
to 800 bps which is inside the ladder. To
make an agarose gel it is necessary to have
the agarose and a 1X Tris-acetate-EDTA
(TAE) solution. Calculations indicated that
to obtain a concentration of .8% we needed
.4 grams of agarose to make a 50 ml agarose
gel. We had 200 mL of a 10X TAE solution
which was very concentrated. We had to
dilute the TAE solution down to a 1X
solution which calculations were necessary
for it. To calculate the dilution we did so in
the following manner:
10X TAE * V
1
=1X TAE * 50mL
The calculations showed that to obtain
50mL of a 1X stock solution of TAE we
needed 5mL of a 10X stock solution of
TAE, and bring to volume of 50mL with
dH
2
O. We measured 5mL of the 10X stock
solution, brought to volume of 50mL in a
graduated cylinder, pour that into a
Erlenmeyer flask, and added the .4 grams of
agarose into the flask. After mixing the
elements the flask goes to be heated in a
microwave for about one minute or until the
substances is easily seen clear and doesn't
have parts of solidified agarose. Let it cool
for a moment, add 1 micro liter of ethidium
bromide, and later pour the contents of the
flask into the gel box. The gel box should be
assembled right now, using the pressure of
the rubber bands, lock the gel box tight so
that no liquid will leak out. With the gel now
inside the gel box add the 10-well comb and
let the gel sit for another 40 minutes or until
it solid. After the gel is solid switch the gel
box from being locked to have its sides open
with its wells being closer to the positive
pole than the negative.
Sample Preparation for Gel
While letting the agarose gel cool and the
digestion tubes incubate. Use the 10X TAE
to make 280mL of a 1X TAE solution.
Again, we calculated the following:
10X TAE * V
1
=1X TAE * 280mL
From the calculations we gathered that to
make a 280mL TAE buffer, for the gel
electrophoresis to run with, we needed
28mL of the 10X TAE solution. We
extracted the 28mL of the 10X TAE and
measured it with a graduated cylinder. We
brought to volume of 280mL by filling the
remaining volume with dH
2
O in order to
dilute the concentrated solution to a 1X TAE
buffer. After the gel is fully solidified pour
the buffer solution into the gel
electrophoresis box until the gel fully
submerged by it, careful not to overflow
however.
Assuming one hour worth of incubation has
transpired already, we removed the tubes
from their incubation bath and set to spin in
the centrifuge for 5 seconds or until the
substances are concentrated together at the
bottom of the tube.
We added 4 micro liters (uL) of ethidium
bromide into each of the digestion tubes
which was calculated from the following
manner:
5X Dye * V
1
=1X Dye * 20uL
After vortexing and centrifuging it again to
homogenize the substances, we extracted
24uL of each one of the digestion tubes and
inserted them inside the already solidified
wells of the agarose gel. In the following
order:
Ladder EcoRI BglI HindIII EcoRI+HindIII Control Ladder
Chart 1.2 Chart 1.2: The order in which the gels have been inserted into the gel and in correlation to Picture
1.1.

Since there are too many to have a space
between them we placed them one after one
with no spaces until the final ladder.
We inserted the ladder (NEB 1K Ladder
N3232L New England BioLabs, all rights
reserved) in 5uL at each of the sides of the
gel.
Close the gel and plugged in the cables, ran
the gel at 140 volts for one hour or until the
bands reach 4 to 5 cm of length.
After it has reached 4-5 cm or has been over
an hour. We removed the gel and brought it
to a UV Light chamber with a camera
incorporated to it. Took three pictures
determined it by underlining the ladders and
underlining the strands of the different cuts.
Measured the distance from the wells to the
cuts in millimeters using a ruler and then
used Excel 2007 (Microsoft Office 2007 -
Blue Edition, All Rights Reserved) to find
the exponential curve and the base pair
results.
In order to predict our findings of the gel,
we used the NebCutter V2.0
(tinyurl.com/NEBcutter) program to obtain
predictions for the pAMP, pBLU, or
pKAMP plasmids. We predicted with the
HindIII, BglI, EcoRI separated, and
HindIII+EcoRI together. We took the
plasmid sequences from Cold Spring Harbor
Laboratory webpage to use it on the
NebCutter V2.0 Webpage.
(http://www.dnalc.org/resources/plasmids.ht
ml)

Results and Conclusions
The concentration was 150ng/ml.
The picture shows that there is still DNA in
the wells which means that there wasn't
enough time for fully digestion. Which
would probably mean it requires more time
for it to fully digest. Also the ladders do not
look fully defined, the reason for that
Picture 1.1: 3rd Gel Picture. From left to right. Ladder - EcoRI - BglI - HindIII - EcoRI + HindIII - Control - Ladder
These pictures are already analyzed by measuring ladder distances and employing an exponential equation in order to find its
specific base pair fragments.
Graph 1.1: This graph shows the standard curve line and the experiments ladder as well as the other fragment
cuts and where they stand.
behavior is unknown as of right now. But it
may deal with the voltage or the lack of
proper digestion of enzymes. Which would
definitively mean to give it more time to
digest next attempted experimentation.
By comparison between pAMP, pKAMP, and
pBLU. At first at a quick glance it was likely it
was pBLU. For a bar at the bottom of the BglI
column which I thought it represented a base
pair lower than 1000 base pairs. Which
would've meant that according to the ladder it
would've been pBLU.


However under further inspection and analyze, I've
found that that wasn't the hoped base pair. Instead it
resembled more to pAMP. This was due to more
resemblance in multiple different base pairs in
comparison to pBLU where there were almost as many
comparisons but they were all pointing at BglI #2, #3;
and HindIII #2. Unlike pAMP resemblance which had
more similarity with BglI #2, #3, #4; EcoRI #1; and
EcoRI+HindIII #3. Out of which I can safely establish
that this plasmid, 5212B58, is in fact pAMP.
However though, extra experimentation with
more careful action may give us deeper insight
into why is it pAMP. For the new
experimentation, careful regulation of voltage
and application of proper buffer along with
extra digestion time may give this experiment
more defined, clearer results.
Chart 1.3: Results from the gel electrophoresis experiment inside an Excel spreadsheet.






y = 112,045.78703259e
-0.11847982x

R = 0.99926133
0
2000
4000
6000
8000
10000
12000
0 10 20 30 40 50
Ladder
BglI
EcoRI
HindIII
EcoRI-HindIII

Band Migration
(mm) Ladder Size (bp)
Ladder 20.5 10000
Ladder 22.6 8000
Ladder 24.5 6000
Ladder 26.2 5000
Ladder 28.1 4000
Ladder 30.6 3000
Ladder 33.6 2000
Ladder 36.2 1500
Ladder 40.1 1000
Ladder 45.8 500
BglI (#1) 22 8269.5
BglI (#2) 28 4062.3
BglI (#3) 28.9 3651.5
BglI (#4) 38.2 1213.3
EcoRI (#1) 26.8 4682.9
HindIII (#1) 21.9 8368.0
HindIII (#2) 26 5148.4
EcoRI-HindIII (#1) 26.5 4852.3
EcoRI-HindIII (#2) 30.2 3130.3
EcoRi-HindIII (#3) 33.1 2220.1
Chart 1.4: Predicted results to compare with the gel electrophoresis results.
References

1. Wikipedia, 2014. Restriction Enzyme. Internet:
http://en.wikipedia.org/wiki/Restriction_enzyme
2. Wikipedia, 2014. Plasmids. Internet:
http://en.wikipedia.org/wiki/Plasmid
3. Sinkovics, J; Harvath J, Horak A. (1998). "The Origin
and evolution of viruses (a review)". Acta
microbiologica et immunologica Hungarica (St.
Joseph's Hospital, Department of Medicine,
University of South Florida College of Medicine,
Tampa, FL, USA.: Akademiai Kiado)
4. Lederberg J (1952). "Cell genetics and hereditary
symbiosis". Physiol. Rev. 32 (4)
5. Kandavelou K; Chandrasegaran S (2008). "Plasmids
for Gene Therapy". Plasmids: Current Research and
Future Trends. Caister Academic Press
6. Lipps G (editor). (2008). Plasmids: Current Research
and Future Trends. Caister Academic Press
7. Thomas, Christopher M; Summers, David (2008).
Bacterial Plasmids
8. Pingoud A, Alves J, Geiger R (1993). "Chapter 8:
Restriction Enzymes". In Burrell M. Enzymes of
Molecular Biology. Methods of Molecular Biology 16.
Totowa, NJ: Humana Press.
9. Comrie, A.C., 2005: Scientific Report Writing.
Internet:http://geog.arizona.edu/~comrie/geog
230/report.htm>



























Fragment Length (bps)
Restriction Enzyme pAMP pKAN pBLU
BglI 3263,
1118,
158,
4381,
1276,
3421
3139,
794,
261,
3933,
3400,
1055
2121,
1740,
1576,
3861,
3316,
3697
HindIII 4539 4194 5437
EcoRI 4539 4194 5437
HindIII+EcoRI 2635,
1904
2312,
1882
5386,
51