Plasmid Identification: Coded 5135E31
Jessi Honeycutt
Introduction
To identify the unknown plasmid,
Plasmids are small, circular double-stranded
restriction enzymes and gel electrophoresis
DNA molecules. Often used as a vector,
were used to compare with a predicted cut
Plasmids carry genes that can be
transmitted from one gene to another.
sit. It was necessary to have a basic
knowledge of understanding how each
restriction enzymes and gel electrophoresis
Restriction Enzymes are enzymes that cut
are used together in the lab. Biotechnology
DNA molecules at specific cut sites, such as
lab techniques use this type of experiment
a base pair sequence.
as a basis for other types of labs. By
Gel Electrophoresis is the process of using
determining an unknown plasmid, one is
electricity to separate DNA molecules by
better off understanding the plasmids uses.
size through a block of agarose gel solution.
Because of the electric field, and DNA’s
ability to run to positive, the samples are
separated according to size, the smaller
molecules “moving” farther than molecules
of a larger size.
Methods
The three options of plasmids for this lab
were pKAN, pAMP, or pBLU. The plasmids
were kept in a 1.5 ml centrifuge tube, and
each tube was marked with only a simple
The goal of this experiment was to
code. The unidentified plasmid I received
determine an unidentified plasmid.
was code named 5135E31.
All reaction buffers, restriction enzymes,
The first step was preparing the DNA
and the plasmids were manufactured by
samples with plasmid DNA, buffer, dH20
New England Biolabs (NEB). The marker
and restriction enzymes. Three micro
ladder was NEB 1Kb ladder, also
centrifuge tubes were needed, labeled
manufactured by New England Biolabs.
control, E (single digest), and N+P (double
The concentration of this plasmid could
have been found in multiple ways, such as
spectrophotometry, but the instructor
simply gave us out of a total of 400 ng, the
plasmids were 150 ng/µl each. To find the
concentration in a total of 20 µL per micro
centrifuge tube, the equation used was 150
ng/µl · x µL = 400ng. X was calculated to be
3.3 µL, used later to determine the
concentration of each individual DNA
sample.
digest). The total volume of each tube
needed to be 20 µL. As shown above, the
starting concentration per all individual
tubes was 3.3 µL. Each tube also received 2
µL of Buffer 3, calculated by the instructor.
To add dH20 each tube was simply
calculated to a total of 20 µL, such that C
received 14.7 µL, E received 13.7 µL, and
N+H received 12.7 µL of dH20. After the
plasmid, buffer, and dH20 were in place,
the restriction enzymes were added. E
received 1 µL of EcoRi restriction enzyme,
The enzymes chosen for the first attempt
were EcoRi in buffer 3 for the single digest,
and NdeI and PstI in buffer 3 for the double
digest.
and N+P received 1 µL of NdeI restriction
enzyme and 1 µL PstI restriction enzyme,
with each measurement being calculated by
the instructor. All three tubes were then
incubated for 1 hour at 37 degrees Celsius
The third step was to run the gel after the
in a dry bath.
samples have finished incubating and the
The second step was preparing the Agarose
Gel. The optimum base pairs for a 0.8%
Agarose solution is 800 – 12,000 bp. To
make a 50 ml 0.8% Agarose solution, it is
calculated that 0.4 g of agarose solid is
needed. 1x TAE is also needed. To dilute 1x
from a 10x stock solution, simply solve for
the unknown volume in the given equation:
50 ml · 1x = (unknown Volume) · 10x, and
gel is solidified. The gel is first covered with
280 ml of 1x TAE buffer, following the
above equation and substituting 280 ml for
50 ml. Next 4 µL of 6x loading dye are
added to each tube. The gel is loaded with 5
µL of NEB 1Kb ladder. And the samples are
each loaded into separate wells, as shown
in Figure 1. The gel is run at 140 volts for 1
hour.
bring to volume 50 ml with dH20. A 0.4g of
The final step was to record the images of
solid is weighed out and along with 50 ml of
the gels and to create a standard curve.
1x TAE, is microwaved in an Erlenmeyer
After the gel was run, the gel was taken to a
flask for 45 seconds, and then in 15 second
UV imaging machine, where a photograph
increments, until all fragments have
was taken of the bands of the DNA, as
become clear. The mixture is put in the Gel
shown in Image 1. A standard curve was
Electrophoresis gel box and allowed to
then created using Microsoft Excel, as
solidify with a 10-well comb, about 30-40
shown in Table 1.
minutes.
Enzymes chosen for the second attempt,
NEBcutter1 and 2. Predicted cuts are shown
EcoRi in buffer 3 for the single digest, and
below.
NdeI and PstI in buffer 3 for the double
Fragment
Fragment
Sizes (bp)
sizes (bp)
All steps were repeated as mentioned
with
with
above, except that the plasmid
EcoRI
NdeI+PstI
digest.
concentration was reduced to 1 µL per
pAMP
4539
3134, 1405
tube, and the incubation period was
pKAN
4194
2635, 923,
extended to 1 hour and 30 minutes.
Enzymes chosen for the third attempt, BgII
636
pBLU
5437
2998, 1316,
926
in buffer 3 for the single digest, and NdeI
and HIndIII in buffer 3 for the double digest.
All steps were repeated in mentioned above
Fragment
Fragment sizes
Sizes (bp)
(bp) with
with
NdeI+HindIII
with 1 µL per tube, and an incubation
period of 1 hour. Results shown in Image 2
and Table 2.
BgII
Predicted Results
All predicted enzyme cuts of pKAN, pBLU,
and pAMP, were taken directly from
pAMP
3263, 1118
2421, 2118
pKAN
3139, 794
2098, 2096
pBLU
2121, 1740,
3006, 2421
1576
Results
The starting concentration of the plasmid was 150 ng.
Image 1: Gel Run, First Attempt. Left to Right: Ladder, Control, EcoRi, NdeI+PstI, Ladder
Image 2: Gel Run, Third Attempt. Left to Right: Ladder, Control(not shown), BgII, NdeI+HndIII,
Ladder
45
First Attempt
40
35
y = 39.679e-0.076x
R² = 0.9229
Distance Traveled (mm)
30
25
20
15
10
5
0
0
2
4
6
DNA fragment size (bp)
Table 1: Standard Curve, First Attempt.
Dna
fragment Ladder
size
Migration
10
20
8
21
6
25
5
26
4
29
3
33
2
37
1.5
40
1
45
8
10
12
45
Third Attempt
40
Distance Traveled (mm)
35
y = 39.679e-0.076x
R² = 0.9229
30
25
20
15
10
5
0
0
2
4
Table 2: Standard Curve, Third Attempt.
Dna
fragment Ladder
size
Migration
10
20
8
21
6
24
5
26
4
28
3
30
2
34
1.5
40
1
6
DNA fragment size (bp)
8
10
12
Conclusion
Based on the data, it is not possible to identify the unknown plasmid code named 5135E31.
Based on the experiments done three times, perfectly and differently, the conclusion that I
have reached is that the plasmid is not correctly functioning. The plasmid did indeed contain
DNA, but it did not digest, for unknown reasons. The reason could have been Insufficient lysis,
lysate was incorrectly prepared, or a malfunction in preparing the plasmid. Because there were
6 different enzymes testing the plasmid, and there was no digestion for any of them, there is
evidence to be seen that it was the plasmid that was inadequate. If I were to repeat this lab, I
would compare my plasmid with another, the plasmid that it was supposed to be. I would use
the same enzymes and buffers, but compare the results of the previous runs with the new. The
purpose of this would be to pinpoint the precise reason the plasmid did not run correctly.