BCH 323

NYARUGWE VICTOR.C : 201515561

PRACTICAL 2 : ISOLATION OF PLASMID

DNA

DATE PERFORMED : 13/09/2017

Title: Isolation of plasmid DNA
ABSTRACT
The use of plasmid DNA is the keystone of DNA analysis because it allows easy
manipulation and maintenance of defined heterologous DNA fragments. The
Escherichia coli bacterial system is very versatile, allowing rapid DNA replication and
informed gene manipulation. Plasmids may be isolated by a variety of methods many
of which rely on the differential denaturation and reannealing of plasmid DNA
compared to chromosomal DNA in this study we used differential denaturation
because of bit harsh conditions which were performed. This method invariably
involves three steps which are growth of the bacterial culture, harvesting and lysis of
the bacteria purification of plasmid DNA.
In this study no restriction enzyme was used, this enzyme is required in the
breakdown of double-strand in DNA at specific sites forming fragments prior to
electrophoresis. This explains why the was no separation of the bands on the other
gel and why the bands on another was too broad and wide such that its size cannot
be determined using a base pair ladder.

INTRODUCTION
The current genetic revolution is actually the result of a long history of remarkable
scientific achievements. In the late 1800s and early 1900s, Gregor Mendel and his
successors determined that heredity was controlled by discrete factors (that we now
call genes). Early cell biologists found a strong correlation between the behaviour of
genes and the behaviour of cellular structures called chromosomes. Biochemists
determined that chromosomes were made of both DNA and protein. Currently,
sophisticated techniques are being exploited for isolating DNA, cloning genes, and
determining how genes function. To comprehend fully these topics, one must
understand some of the basic principles of DNA analysis such as electrophoresis
(Daniel & William ,2012).

Agarose gel electrophoresis is a process that undertakes biochemistry and

molecular biology understandings to identify and analyse DNA and RNA strands.
This is done by separating the genetic material by its size. The genetic material is
placed in the solidified wells of the agarose (a linear polymer composed of
alternating isomers of the sugar galactose) at the cathode end. The negatively
charged nucleic acid molecules move through the agarose matrix with the assistance
of an electric field (electrophoresis). This is because genetic material is negatively
charged, and will move towards the anode when current is passed through (Maniatis,
et al.,1989).

The shorter molecules migrate faster than the longer molecules. The use of
electrophoresis buffer in the making of the agarose gel is to establish a constant pH
and to provide ions to support the conductivity. If instead water was used, then the
genetic material will not migrate during the electrophoresis. The amount of voltage
used is crucial to the migration of the genetic material. When increasing voltage is
applied to the gel, larger fragments migrate proportionally to that of the smaller
fragments. Thus, the voltage applied is usually 5 volts per centimetre to the gel. The
gel is then immersed in ethidium bromide, a fluorescent dye that covalently binds
(intercalates) between the bases of nucleic acid. Then UV light is passed through the
gel to make the genetic material visible (Starr, et al.,2011).

AIM
To isolate DNA from plasmid culture and to observe its ability to resist an antibiotic
(ampicillin) as well as determining its size using a DNA ladder.

MATERIALS AND METHODS

RESULTS
DISCUSSION
Plasmids are minute genetic elements that replicate separately from the
chromosome. Majority of plasmids are in the form of double-stranded DNA (dsDNA)
and may either be circular or linear. Plasmid DNA is usually employed in
recombinant DNA technology to clone a specific segment of DNA resulting most
advantageously in that large quantities of these segments can be prepared.
Moreover, plasmid DNA have inherent antibiotic resistance genes and are used to
introduce genes into cells by transformation. DNA is found in a variety of different
sizes and configurations in different organisms. The chromosomes of higher
organisms, such as man, contain long, linear molecules of DNA. In bacteria, the
chromosomes are circular, as are the DNA molecules contained in these
chromosomes. Although these circular DNA molecules are usually broken into linear
fragments during purification, some bacteria also possess smaller extrachromosomal
circular DNA molecules that are easier to isolate without breaking. These
extrachromosomal DNA molecules are called plasmids and contain genes that are
not essential to the bacteria but confer specialized functions such as resistance to
antibiotics (Birnboim & Doly,1979).
In this practical the plasmid culture was prepared with an antibiotic selection
(ampicillin), antibiotics are generally defined as agents that kill bacteria, or inhibit
their growth. Before the culture was left for overnight the bacterial culture was yellow
and after 24 hours it was cloudy which was an indication of some resistance towards
ampicillin. Antibiotic resistance occurs when bacteria change in some way that
reduces or eliminates the effectiveness of drugs, chemicals, or other agents
designed to cure or prevent infections. The bacteria survive and continue to multiply
causing more harm. If a microbe is resistant to many drugs, treating the infections it
causes can become difficult or even impossible. Someone with an infection that is
resistant to a certain medicine can pass that resistant infection to another person. In
this way, a hard-to-treat illness can be spread from person to person. In some cases,
the illness can lead to serious disability or even death (Holmes & Quigley,1988).
From the results of the gels obtained, on the other gel there is no separation of DNA
into bands whereas on the other gel the bands are too broad and wide such that they
cannot be measured using a DNA ladder. The explanation to this is that we used
DNA as a whole however, similar experiments that were carried out involves the use
of restriction enzymes, these enzymes are also known as restriction endonucleases,
recognize short DNA sequences which are often palindromic. These enzymes cleave
double-stranded DNA at specific sites within or adjacent to their recognition
sequences. When a restriction enzyme is used to cleave the DNA fragments, various
sizes of fragments are formed and the methylated cytosine produces larger DNA
fragments. Therefore, it is noticeable that when electrophoresis is being conducted,
smaller DNA fragments travels at a faster rate and larger DNA fragments travel at
lower rate (Brown ,2008).
REFERENCES
Birnboim H and Doly J ,1979, A rapid alkaline extraction procedure for
screening recombinant plasmid DNA. Nucleic Acids Res, page 99-103.

Brown T ,2008, Introduction to Genetics: A Molecular Approach, volume 1,

page 203-205.

Daniel Fand William R,2012, Genetics - The Continuity of Life, page 88-92.

Holmes D and Quigley M ,1988, A rapid boiling method for the preparation of
bacterial plasmids. Anal. Biochem. 114, 193

Starr L, Starr C, Taggart R, Evers A ,2011, Cell Biology and Genetics, Volume
1 International Edition, 13th edition, page 120-120

Maniatis T, Fritsch E, Sambrook J ,1989, Molecular Cloning: A Laboratory

Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.
368,369.