Plasmid Identification Project

April 30, 2014

Plasmid Identification Project
Biotech 1015
Whitney Morris
April 30, 2014








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Introduction
What is a plasmid? A plasmid is a circular piece of DNA that is smaller than a
chromosome and is used to carry genes into a cell. Plasmids are very useful in finding
and isolating a gene, because they replicate independently. When identifying a plasmid a
restriction enzyme is used. A restriction enzyme is an enzyme that cuts DNA into a
specific sequence. You will then run the plasmid and enzyme through a gel
electrophoresis. Gel electrophoresis will separate the DNA, of the plasmid, using an
agarose gel. This separation is based on size, the DNA runs from the black electrode
(negative) to the red electrode (positive). Understanding the information explained above
will allow you to understand the basis of this experiment.
The goal of this experiment was to identify an unknown plasmid by using three
restriction enzymes and then running a gel to help identify the unknown plasmid.
Identifying the plasmid demonstrates the skills learned in Biotech 1015. The strategy of
my experiment was to use the restriction enzymes BamHI, PstI and Pvul to cut and
identify my unknown plasmid. By using those restriction enzymes, which run in buffer
three, allowed me to go through a process of elimination which permitted me to identify
the plasmid.
Methods
The code for my unknown plasmid was: 7936A65. For this assignment I was
given the concentration of my plasmid due to the limit on time.. The concentration of my
plasmid was, 150ng/ 1L, which then allowed me to find the amount of unknown
plasmid, dH2O, each enzyme, and buffer 3.The amounts can be found in the appendix in
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table 1. The DNA latter used was a NEB1Kb from the New England Bio labs, as well as
the restriction enzymes and buffers. The digest recipes I used were a single digest with
BamHI, and a double digest with PstI + Pvul. My sample incubated for one hour at 37
degrees Celsius in a hot plate.
The gel I made was a 1% agarose gel in 50 mL of 1x TAE. I had to diluted a 10x
TAE to a 1x TAE by adding 5 mL of the 10x TAE to 45 mL of dH2O. I used 0.5 grams of
agarose and added that to the beaker containing the 50 mL of the dilute 1x TAE. I then
heated the solution in the microwave for four 30-second intervals, until the solution
became clear. After heating the solution I added 5 L of ethidium bromide to the
solution. Once the solution had cooled enough I poured the solution into the gel box and
allowed it to sit for 30 minutes. Once my sample had finished incubating I added 4 L of
6x loading dye to the solution. The loading dye allows the sample to be seen as it runs
through the gel. I then inserted the 1 KB ladder into the gel on both sides of the gel. I then
inserted each sample into a well. I ran the gel at 140 volts until the bottom dye band was
between the four and five, which took roughly one hour. The buffer used in the gel box
was 280 mL of a 1X TAE. After my gel was finished running I took a picture of it on the
UV imaging system, which can be seen in table 2.
I was able to determine the sizes of my DNA from the latter as well as from data
collected from the NEB cutter website, which provided the sizes of the bands that the
enzyme would cut. By using the NEB cutter website, I was able to get an idea of what
each gel would generally look like depending on where the sample was cut in the gel. I
then made a standard curve graph based on the distance from the middle of the wells to
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the middle of the band. The standard curve allowed me to gain a greater understanding on
the values of the bands in the gel.
Results and Conclusion
The concentration of my DNA was 150 ng/ 1 L, I needed 2 L of 10X
NEBbuffer 3, the total volume of the pCR tube needed to be 20 L. With each of my
digests and the control, they contained either 1 L of the enzymes needed in that digests
or none at all. The PCR tube had 1 L or no enzymes, which allowed me to find the
remaining amount of dH
2
O needed for the pCR tube.
The data for the amount shown to the side is the gel lists the DNA Latter sizes.
The latter was run in the first well on the left and the second to last well on the right. The
third well from the left was my control sample, fifth one in, was the sample containing
BamHI, and the seventh one in contained PstI + Pvul. By creating a standard curve,
which is shown in the appendix labeled table 3, it allows a general idea of where the cuts
on the DNA should be located. Listed below table 3 in the appendix, in the table are all of
the possible base pair sizes, which would be on the cut DNA for the plasmids. By looking
at those cut sizes and where they are located on the gel, I was able to determine which
plasmid I was given.
Based off of the information and from the data collected I was able to come to the
conclusion that my plasmid was pBLU. I was able to come to this conclusion because
when I compared my gel to where the cuts on the other plasmids the pBLU was the only
possible reason for a cut above the ladder, roughly at 6046. My sample wasnt incubated
enough so the DNA wasnt cut completely causing the DNA to be above the ladder.
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When attempting a second test on my unknown plasmid, my samples were not incubated
at the right temperature for the right amount of time. Figure 2 shows that none of the
DNA was digested, so none of the DNA went farther down then the ladder. Due to the
lack in data from the gel, I was not able to draw any conclusions from the gel or create a
standard curve from the gel. Based on the results from my first test, pBLU is able to
account for all of the cuts in the PstI + Pvul test; pAMP and pKAN could only match two
bands and still had a distance close to 150 between the actual band and where it was
supposed to cut based on the data. Looking at all the data and by using the R-value off
my Standard Curve I concluded that my plasmid is pBLU.


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Appendix
Table 1:
Tube Unknown 10x NEB
Buffer
dH2O BamHI PstI Pvul Total
volume
Control 2 L 2 L 16 L ---------- ---------- -------- 20 L
BamHI 2 L 2 L 15 L 1 L ---------- --------- 20 L
PstI + Pvul 2 L 2 L 14 L ---------- 1 L 1 L 20 L






Figure 1

First row on left: 1KB Ladder
Size of bands:
10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5
Second row: Control
Third row: BamHI
Fourth row: PstI + Pvul
Fifth row: KB Ladder
Size of bands:
10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5

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y = 6E+07x
-2.859

R = 0.99966
0
2000
4000
6000
8000
10000
12000
0 10 20 30 40 50
B
a
n
d

M
i
g
r
a
t
i
o
n

(
m
m
)


Ladder Size (bp)
Standard Curve
Figure 2

First row on left: 1KB Ladder
Size of bands:
10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5
Second row: Control
Third row: PstI+ Pvul
Forth row: BglI
Fifth row: BamHI
Sixth row: HindIII
Seventh row: 1KB Ladder
Size of bands:
10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5

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Band Migration (mm) Ladder Size (bp)
Ladder - Band 1 21 10000
Ladder - Band 2 23 8000
Ladder - Band 3 25.2 6000
Ladder - Band 4 27 5000
Ladder - Band 5 29.2 4000
Ladder - Band 6 32 3000
Ladder - Band 7 37 2000
Ladder - Band 8 40.8 1500
Ladder - Band 9 47.5 1000
Control - Band 1 12.2 47016
BamHI - Band 1 12.2 47016
BamHI - Band 2 19 13249
BamHI - Band 3 25 6046
PstI+PvuI - Band 1 28.5 4157
PstI+PvuI - Band 2 35 2310
PstI+PvuI - Band 3 39 1695
PstI+PvuI - Band 4 42.6 1317
PstI+PvuI - Band 5 46.4 1032
PstI+PvuI - Band 6 50.5 810

Table 4: Base pair cuts
Circular Sequence: pAMP
# of cuts Enzymes Coordinates Length (bp)
1 BamHI-BamHI 1018-1017 4539

1 PvuI-PstI 3157-946 2329
2 PstI-PvuI 947-2260 1314
3 Pvul-Pvul 2261-3156 896



Circular Sequence: pKAN
# of cuts Enzymes Coordinates Length (bp)
1 BamHI-BamHI 2096-2095 4194

1 Pvul-PstI 2812-771 2154
2 PstI-PstI 772-1694 923
3 Pvul-Pvul 2240-2811 572
4 PstI-Pvul 1695-2239 545
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Bibliography
BioLabs, N. E. (2003). Double Digest Finder . Retrieved April 30, 2014, from New
England BioLabs : https://www.neb.com/tools-and-resources/interactive-
tools/double-digest-finder

BioLabs, N. E. (2003). NEBuffer Performance Chart. Retrieved April 30, 2014, from
New England BioLabs: https://www.neb.com/~/media/NebUs/Files/nebuffer-
performance-chart-with-restriction-enzymes.pdf

Laboritory, C. S. (n.d.). DNA Learning Center . Retrieved April 30, 2014, from Cold
Spring Harbor Laboratory : http://www.dnalc.org/resources/plasmids.html

Vincze, T. P. (2003). BioLabs . Retrieved April 30, 2014, from NEBcutter:
http://tools.neb.com/NEBcutter2/index.php


Circular Sequence: pBLU
# of cuts Enzymes
Coordinates
Length (bp)
1 BamHI-BamHI 3161-3160 5437

1 Pvul-Pvul 4821-1363 1980
2 PstI-PstI 3380-4695 1316
3 Pvul-Pvul 2297-3022 726
4 Pvul-Pvul 1817-2296 480
5 Pvul-Pvul 1364-1816 453
6 PstI-PstI 3183-3379 197
7 Pvul-PstI 3023-3182 160
8 PstI- Pvul 4696-4820 125