Plasmid Identification Experiment Analysis

Plasmid Code: 5832D84

By: Michael Shields
In this experiment, I, Michael Shields, identified an unknown plasmid using restriction
enzymes and gel electrophoresis. A plasmid is a DNA molecule that can be manipulated easily.
1
A restriction enzyme is simply an enzyme capable of cutting DNA in a specific location.
2
Gel
electrophoresis is a method of cutting DNA, according to size. Through this process the
molecules to be separated are pushed by an electrical field through a gel that contains small
pores.
3
The DNA then travels at different speeds according to its size, (small pieces go faster
and farther while big pieces go slower, and not as far.) The goal of this experiment was to
analyze and evaluate the type of plasmid that I was given (pAMP, pKAN, or pBLU), as well as to
do so by utilizing my own methods and processes. This goal is particularly important to
understanding biotechnology lab techniques because it was my specific techniques and
research, instead of simply copying someone elses work. My strategy during this experiment
was to utilize a very specific set of restriction enzymes so as to make data analysis easy.
The methods for this project were fairly simple. I first ascertained the restriction
enzymes that Id be using, after that I mixed them with the plasmid assigned to me (code name:
5832D84), according to my recipe. My recipe for this plasmid mixture was 3.3microliters of the
DNA, 1 microliter of the enzyme (either 1microliter of EcoRI, or 1microliter of Ndel and PstI),
and adding 2 microliters of buffer (3.1) and bring the mix to a volume of 20 microliters with
deionized water (dH
2
O). The concentration of the solution was given (150ng/ml). Next, I
incubated the mixture in a heat bath (about 30
o
Celsius) for one hour, while that was happening
I mixed together an agarose gel (0.8% agarose, made by combining 0.4g of agarose with 50mL
of dH
2
O and bringing to volume 150mL). After the plasmid had incubated I ran the DNA in a gel
electrophoresis chamber, with two ladders, for 1.5 hours, at 130 volts. The source of my ladder
was New England Biology Labs, as well as my enzymes and original plasmid. I then removed the
gel, photographed it, and analyzed the data. To analyze the data, I measured the lengths of the
ladder cuts and compared those to the lengths of my own DNAs cuts. I was able to predict this
contrast with the use of an online software
4
.
The starting concentration of my plasmid was 150ng/ml. Below, are the two pictures of

my gel, and here is a graph of my Ladder Migration, it shows that I have my cuts (from the
plasmid) on track with how my ladder was supposed to cut.

This is a table showing my results from (
4
) this is the data that was used to find what plasmid
was being analyzed, as well as my plasmid (X) and allowed for the results to be found.
Single Digest Double Digest
Plasmid Enzyme: EcoRI Plasmid Enzyme: Ndel PstI Both (Ndel & PstI)
pAMP 4539 pAMP 4539 4539 3134, 1405
pKAN 4194 pKAN 4194 3271, 923 2635, 923, 636
pBLU 5437 pBLU 5437 3725, 1134, 197 2498, 1316, 926, 197
y = 30.483e
-0.117x

R = 0.9704
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12
Ladder Migration (mm)
Ladder Migration (mm)
Expon. (Ladder Migration (mm))
(X) 5300 (X) N/A N/A Inconclusive
It was concluded that the unknown plasmid was pBLU, this was found by looking into the data
above and reasoning that the closest one must be the correct one. The double digest had no
data that was relevant to my ladders, and as a consequence of such has been categorized as
inconclusive.
















Sources and Footnotes:
1. Guruatma Ji Khalsa, 2010, Mama Ji's Molecular Kitchen. Internet:
http://askabiologist.asu.edu/plasmids.
2. Guruatma Ji Khalsa. 2010 "Mama Ji's Molecular Kitchen." Internet:
http://askabiologist.asu.edu/restriction-enzymes.
3. 2014. http://www.nature.com/scitable/definition/gel-electrophoresis-286
4. Vincze, T., Posfai, J. and Roberts, R.J.
NEBcutter: a program to cleave DNA with restriction enzymes
Nucleic Acids Res. 31: 3688-3691 (2003) http://tools.neb.com/NEBcutter2/