Identification of Unknown Plasmid

Teresa M. Huff (1T)

Introduction
In the experiment I was given an unknown plasmid and was to determine if the Plasmid was
pAMP, pKAN, or pBLU. According to Biology Online the definition of a plasmid is, A linear or
circular double-stranded DNA that is capable of replicating independently of the chromosomal
DNA (Biology Online, 2009, para. 1). Plasmids have been used for DNA-Cloning experiments
and has genes coding for antibiotic resistance. To determine the unknown plasmid I performed
a restriction digest with different enzymes. Restriction enzymes are proteins that cut DNA to
make fragments. Then I proceeded by running a gel of my restriction digests. Gel
electrophoresis uses an electrical charge to separate the different sizes of DNA and RNA
fragments. To determine the fragment sizes I used a standard curve and compared them to a
virtual digest I performed of the three plasmids. To be able to identify an unknown plasmid is
important to my understanding of lab techniques to show I understand and can do these things
in a lab.

Methods
The unknown plasmid that I had chosen had the code: 2435D81. Before receiving the plasmid I
had chosen what restriction enzymes I would use. I used the New England BioLabs NEBcutter
V2.0 to perform a virtual digest of the three plasmids where we had received the sequences
from the DNA Learning Center. I chose an enzyme, from the options available in our lab, which
had fragment sizes that were easy to differentiate between the plasmids. The enzymes I chose
were PstI and for a double digest I used BglI and BamHI. From the New England BioLabs page on
NEBuffer performance with restriction enzymes it showed I needed to use NEBuffer 3 for my
restriction enzymes. The concentration of my plasmid was 150 ng/mg which was given by my
instructor who wouldve used the Nanodrop to figure out the concentration of the plasmid. I
labeled three 1.5 mL tubes: Control, PstI, and BglI & BamHI. Then calculated the amount of
Plasmid, Buffer 3, and dH
2
O to add to each tube to get a total volume of 20g. For the control I
inserted 3.33L of plasmid, 2L of buffer 3, and 14.67L of dH
2
O. In PstI I inserted 3.33L of
plasmid, 2L of buffer 3, 1L of PstI, and 13.67L of dH
2
O. In BglI and BamHI I inserted 3.33L
of plasmid, 2L of buffer 3, 1L of BglI, 1L BamHI, and 12.67L of dH
2
O.
Test 1
Tube Plasmid Buffer 3 dH
2
O PstI BglI BamHI Total
Control 3.33L 2L 14.67L - - - 20L
PstI 3.33L 2L 13.67L 1L - - 20L
BglI & BamHi 3.33L 2L 12.67L - 1L 1L 20L

Each of the tubes were then flicked to mix and briefly spun to accumulate the liquid at the
bottom. The three tubes were then incubated at 37 degrees Celsius for an hour. I then
proceeded to make the agarose gel for electrophoresis. First by assembling the gel tray, gel box,
10-well comb and power supply. The expected bands were between 10,000 bp and 800 bp so a
0.8% agarose concentration would be needed. To make 50mL of a 0.8% gel I would need 0.4g of
agarose. After measuring 0.4g of agarose solid I poured it in a 250ml Erlenmeyer flask. Then
measuring 50ml of 1X TAE by using the 10X TAE which was made previously. This resulted in
putting 5ml of 10X TAE in the flask and bringing it to a total volume of 50ml with dH
2
O. The
solution was microwaved until the agarose was completely melted. Then 1L of ethidium
bromide was inserted to the solution and lightly swirled in to mix. After letting the solution cool
it was then poured into the gel tray and the 10-well comb was inserted in. It took 20 to 30
minutes to let the agarose solidify. The 10X was used to make 250ml of a 1X TAE to pour into
the gel box. This was done with 25ml of the 10X and brought to 250ml with dH
2
O. After an hour
of incubation the three tubes were then taken out and 4L of loading dye was inserted in and
mixed by pipetting up and down. After the gel had solidified the 250ml of 1X was poured into
the gel box. The ladder used was the DNA 1 kb ladder from New England Biolabs (NEB). 5L of
the ladder was added in well 1 and 9. 24L of the control in well 3, 24L of the PstI in well 5,
and 24L of BglI & BamHI in well 7.
Ladder Blank Control Blank PstI Blank B+B Blank Ladder Blank

The gels were run on a voltage of 140 volts until the dye band was between the 4 and the 5
centimeter mark. After turning off the gel electrophoresis the agarose gel was taken out of the
gel box and placed in the UV Transilluminator machine to take the photo. The DNA sizes were
then determined from the photo by measuring with a ruler in millimeters the distance each
DNA band had gone from the wells and comparing it to the ladder bands which the sizes in bp
were already known.
A second test was then done with the same steps but with different enzymes. The enzymes that
I used for the second test were Xbal, Xhol, PstI, and SacI. For Xbal and Xhol I used buffer 4, for
SacI I used buffer 1, and for PstI I used buffer 3. To ensure I had enough plasmid I only used
1.5L for each tube. I repeated the same steps with incubation and gel electrophoresis.
Test 2
Tube Plasmid Buffer dH
2
O Xbal Xhol PstI SacI Total
Control 1.5L 2L 16.5L - - - - 20L
Xbol 1.5L 2L 15.5L 1L - - - 20L
Xhol 1.5L 2L 15.5L - 1L - - 20L
PstI 1.5L 2L 15.5L - - 1L - 20L
SacI 1.5L 2L 15.5L - - - 1L 20L

Ladder Blank Control Xbol Xhol PstI SacI Blank Ladder Blank





Results and Conclusions
Figure 1






Ladder Blank Control Blank PstI Blank B+B Blank Ladder Blank









Test 1 Band Migration (mm) Ladder Size (bp)
Ladder (Band 1) 19.5 10,000
Ladder (Band 2) 21.2 8,000
Ladder (Band 3) 23.8 6,000
Ladder (Band 4) 25.2 5,000
Ladder (Band 5) 27.8 4,000
Ladder (Band 6) 30 3,000
Ladder (Band 7) 34.5 2,000
Ladder (Band 8) 37.8 1,500
Ladder (Band 9) 42.5 1,000
Ladder (Band 10) 49.2 500
Control (Band 1) 20 8714.79
PstI (Band 1) 23 6475.47
BglI and BamHI (Band 1) 24 5865.11
BglI and BamHI (Band 2) 28 3947.26
BglI and BamHI (Band 3) 33 2406.14
BglI and BamHI (Band 4) 36 1787.86









Figure 2






Ladder Blank Control Xbol Xhol PstI SacI Blank Ladder Blank


















Test 2 Band Migration (mm) Ladder Size (bp)
Ladder (Band 1) 22 10,000
Ladder (Band 2) 23.5 8,000
Ladder (Band 3) 26 6,000
Ladder (Band 4) 27 5,000
Ladder (Band 5) 29.5 4,000
Ladder (Band 6) 32 3,000
Ladder (Band 7) 37 2,000
Ladder (Band 8) 40 1,500
Ladder (Band 9) 46 1,000
Ladder (Band 10) 53.5 500
Control (Band 1) 22 8452.36
Xbal (Band 1) 22 8452.36
Xhol (Band 1) 22 8452.36
pstI (Band 1) 26 5826.67
SacI (Band 1) 22 8452.36
DNA fragment sizes (bp) produced from digestion with
XbaI PstI BglI and BamHI XhoI SacI
pAMP - 4539 2149, 1118, 1114, 158 - -
pKAN - 3271,923 3139, 794, 173, 88 4194 4194
pBLU 5437 3924, 1316, 197 2121, 1740, 1410, 166 - 3495, 1942
Experimental plasmid 8452 6475, 5827 5865, 3947, 2406, 1788 8452 8452

The starting concentration of my plasmid was 150 ng/ml. In my first test I had performed a
restriction digest with the enzymes: PstI, and BglI & BamHI. From the images of my gel that I
had made a standard curve for it had given me the bp sizes. For the PstI it was 6475 and for the
BglI & BamHI it was 5865, 3947, 2406, and 1788. The bp size for PstI did not match any of
predicted fragment sizes. I concluded the reason for this was the DNA did not cut from the lack
of incubation time. The fragment sizes for the BglI and BamHI had two that matched closely
with pBLU and the other two that didnt match. From this data I concluded that my
experimental plasmid was in fact pBLU. I then performed a second digest to confirm if my DNA
was pBLU. The enzymes I chose were XbaI, Pstl, XhoI, and SacI. I had chosen four enzymes to
ensure my conclusion of it being pBLU. The result of this test unfortunately didnt go well. The
fragment sizes I had received again did not match the expected fragment sizes. For the enzymes
XbaI, XhoI, and SacI I had received the same fragment sizes of 8452. It was the same size as the
control which I then concluded they had not cut. The PstI size was 5827 which was close to the
size of the first test. A reason that my DNA had not cut could be that I had not incubated them
long enough. Had I had more DNA I wouldve done a third test using the same enzymes in test 2
but would incubate them for two hours instead of one. Because the results from my second
test could not conclude anything I decided to stay with my first test that my plasmid was pBLU.
References
o Websites
Biology Online, 2009: Plasmid. Internet: <http://www.biology-
online.org/dictionary/Plasmid>.
New England BioLabs, n.d.: NEBuffer Performance Chart with Restriction Enzymes.
Internet: <https://www.neb.com/~/media/NebUs/Files/nebuffer-performance-chart-
with-restriction-enzymes.pdf>.
New England Biolabs, n.d. : NEBcutter V2.0. Internet:
<http://tools.neb.com/NEBcutter2/>.
o Book
Thieman, W.J. and Palladino, M.A., 2009: Introduction to Biotechnology. Pearson
Education, San Fransisco.