DeShayla Tran

Biotechnology 1015
Plasmid Identification
Introduction:
When health is absent, wisdom cannot
reveal itself, art cannot manifest, strength
cannot fight, wealth becomes useless, and
intelligence cannot be applied.
-Herophilus (335 B.C.-280 B.C.)
Biotechnology is typically defined as using
living organisms, or the product of living
organisms, for human benefit by deciphering
predicaments. With the current available
technology, it is possible to cure various
diseases using experimentation, such as DNA
cloning, splicing, etc. In particular, when
cloning DNA, plasmids are used in being that
they contain genes which code for antibiotic
resistance proteins. Plasmids are found
predominantly in bacterial cells and are small,
circular, self replicating DNA molecules.
Restriction enzymes, on the other hand, are
DNA cutting enzymes that are discovered in
the bacteria as well. When functioning, the
enzyme cuts the phosphodiester backbone of
the double-stranded DNA at a restricted
nucleotide sequence; henceforth, creating it to
be imperative in molecular biology. To identify
and separate the various sizes of biomolecules,
gel electrophoresis is used and does so by using
an electrical charge through a semi solid
separating gel matrix, like agarose or
polyacrylamide. In this experiment, all methods
were used to distinguish the type of nucleotide
sequence the given unknown plasmid had
possessed. It is significant to be able to discern
the specific plasmid because, by doing so, it
portrays the amount of knowledge retained
from the biotechnology class in all; depending
on how successful the outcome of the
experiment is. Briefly, by calculating the

amounts acquired for the specific sample and

creating an agarose gel in order to identify the
plasmid, the experiment resulted to be
somewhat effective.
Methods:
In this lab experiment, plasmid 3585A22 had
been obtained. To determine the concentration
of the plasmid, the equation (concentration)
ngL=x amount of ng lead to the numbers in
the chart below.
Plas
mid

Buff
er
3.1

Wate
r

PstI

Pvull

AflII
I

Total

Singl
e
(PstI
)

7l

2l

10l

1l

-----

-----

20l

Dou
ble
(Pvu
l+Af
lIII)

7l

2l

9l

-----

1l

1l

20l

Cont
rol

7l

2l

11l

-----

-----

-----

20l

In order to mark the plasmid, five microliters

of NEB kb ladder was added to the border
wells. For the single digest, PstI had been
added to the digest and for the double, PvuI
and AflIII. For an hour and thirty minutes, the
digests, along with the control, had been
incubated at 37. To create a gel 0.5 grams
of the 1% agarose concentration had been
added due to the fact that
1 gram 100 mL=x 50 mL . To dilute the
10X TAE buffer to a 1X TAE buffer, 5 mL of
the 10X TAE buffer was added to 45 mL of
deionized water. After microwaved, five

microliters of ethidium bromide must be

added to
the solution to expose the plasmid cuts. When
solidified, the gel was ran at a voltage of 140
for about an hour and a half. After it reached
the 5 mark, the gel was removed and set in
the UV transilluminator system to record
images of the gel. To determine the sizes of
the DNA fragments of the gel, each line had
been measured and documented in a chart. In
sum, to predict the fragments that was
produced from each digest, the equation
determined by google excel was used.
Results and Conclusions:
To identify the obtained unknown plasmid,
online charts were created for pAMP, pBLU,
and pKAN for the specific enzymes of the
double and the single digests.
Single (PstI)

Fragment Sizes
(bp)

pAMP

4539

pKAN

3271, 923

pBLU

3924, 1316, 197

Double (AflIIIPvuI)

Fragment Sizes
(bp)

pAMP

4539

pKAN

3271, 923

pBLU

1263, 997, 535,

459, 306, 21

When the UV images were obtained, the

single was very similar to the control; hence,
causing the single to have to be redone.

Ladder-Single-Control-Double-Ladder

Single-Ladder
After measured, separate charts and graphs
were created for each picture.

Band Migration (mm)

Ladder Size (kp)

Ladder (Band 1)

14.0

10.0

Ladder (Band 2)

15.0

8.0

Ladder (Band 3)

16.0

6.0

Ladder (Band 4)

17.0

5.0

Ladder (Band 5)

18.5

4.0

Ladder (Band 6)

20.0

3.0

Ladder (Band 7)

23.0

2.0

Ladder (Band 8)

25.5

1.5

Ladder (Band 9)

29.5

1.0

Ladder (Band 10)

36.0

0.5

Double Digest (Band 1)

11.5

10.96791157

Double Digest (Band 2)

12.0

10.25713827

Double Digest (Band 3)

15.5

6.417148625

16.0

6.0012866

Double Digest (Band 4)

Double Digest Chart

Double Digest Graph

Band Migration (mm)

Ladder (Band 1)

12.0

Ladder Size (kp)

10.0

Ladder (Band 2)

13.0

8.0

Ladder (Band 3)

14.0

6.0

Ladder (Band 4)

16.0

5.0

Ladder (Band 5)

17.0

4.0

Ladder (Band 6)

19.0

3.0

Ladder (Band 7)

20.0

2.0

Ladder (Band 8)

22.5

1.5

Ladder (Band 9)

26.0

1.0

Ladder (Band 10)

30.0

0.5

Single Digest (Band 1)

12.0

9.0406429

Single Digest (Band 2)

19.0

2.86833089

31.0

0.4008092259

Single Digest (Band 3)

Single Digest Chart

Single Digest Chart

Though, the double digest picture was not

accurate in regards to the ladder seeping into
the wells, the single digest had displayed that
the unknown plasmid had a nucleotide
sequence that matched up with pKAN. This
information is precise due to the fact that the r2
from both graphs are close to one. Some
predicaments that had occurred during this lab
was that there was some doubts about if the
ethidium bromide was added in or not, causing
the need to remake the gel and the ladder
seeping into the wells of the double digest, as
stated before. To resolve these issues, next time
the erlenmeyer flask can be marked when
ethidium bromide is added and being more
careful when adding the digest and ladders.
Conclusively, based on the single digest, the
unknown plasmid is defined as pKAN.

References:
Quoteshttp://www.quotessays.com/biotechnology.htm

DefinitionThieman and Palladino, Introduction to Biotechnology, Second Edition