MIC 271- LABORATORY METHOD IN

MOLECULAR BIOLOGY

LABORATARY REPORT

EXPERIMENT: 2
TITLE: PLASMID EXTRACTION

NAME: NUR AMEERA SHAHIRAH BINTI NOOR AZMAN

STUDENT NO: 2018246832
GROUP: AS114 4B1
DATE OF SUBMISSION: 13th APRIL 2020
LECTURER’S NAME: MADAM NUR INTAN HASBULLAH
INTRODUCTION
A plasmid is a special form of DNA found in bacteria. Plasmids are extra
chromosomal genetics elements present in most species of Archae, Eukarya and Eubacteria
that can replicate independently. Plasmids are circular double stranded DNA molecule that
are distinct from the cells chromosomal DNA. In some cases, plasmids are generally not
essential for the survival of the host bacterium. Plasmids specify traits that allow the host to
persist in environments that would otherwise be either lethal or restrictive for growth.
Plasmids have served as invaluable model systems for the study of processes such as DNA
replication, segregation, conjugation, and evolution. Plasmids have been pivotal to modern
recombinant DNA technology as a tool in gene-cloning and as the vehicle for gene-
expression. The isolation of plasmid DNA from bacteria is a crucial technique in molecular
biology and is an essential step in many procedures such as cloning, DNA sequencing,
transfection, and gene therapy.

Agarose gel electrophoresis is a method that commonly used in biochemistry and

molecular biology to isolate a DNA plasmid from bacteria. It is used to separate DNA and
RNA fragments according to length are used to estimate the size and charge of the DNA and
RNA fragments or to protein by size. The aim of agarose gel electrophoresis is to analyse the
plasmid DNA that was extracted. The technique of electrophoresis is based on the fact that
DNA is negatively charged at neutral pH due to its phosphate backbone. The rate of the the
DNA slows down when its moves towards opposite poles because of the agarose. The
agarose buffer gel is a buffer solution that is used to maintain the required pH and salt
concentration.

OBJECTIVE
1. To extract plasmid DNA from bacterial cells.
MATERIALS
Sample
Fresh overnight bacterial solution
Apparatus
Eppendorf tube
Micropipette
Micropipette tips
Vortex
Centrifuge
Spin column
Chemicals
Buffer A1 (Resuspension buffer)
Buffer A2 (Lysis buffer)
Buffer A3 (Neutralization buffer)
Buffer AQ (Wash buffer)
Buffer AE (Elution buffer)

METHODS
A. Plasmid Extraction

1. Pellet cells/ E.coli which are taken from previous experiment were put in a
microcentrifuge tube for 5 minutes at 13000rpm and discarded the supernatant.
2. 150µL Buffer A1 was added and was vortexes to resuspend cells completely.
3. 250µL Buffer 12 was added and was mixed by inverting the tube 5 times.
4. To lyse the cells, the tube was incubated up to 2 minutes at room temperature.
5. 350µL Buffer A3 was added and immediately inverted the tube until the lysate has
turned colourless.
6. The tube was spun at full speed in the centrifuge for 3 minutes to pellet precipitate.
7. A NucleoSpin® Plasmid EasyPure Column was put into a Collection Tube (2mL).
8. The clear supernatant was loaded onto the spin column.
9. The tube was spun at 1000-2000g in the centrifuge and flow-through discarded.
10. 450µL Buffer AQ was added to the spin column.
11. The tube was spun again for 1 minute at full speed and the supernatant was discarded.
The step was repeated again to remove all the liquid.
12. The spin column was put into a new 1.5mL microcentrifuge tube.
13. 50µL Buffer AE was added onto the membrane and incubated for 1 minute.
14. The tube was centrifuged for 1 minute at full speed.

B. Gel Electrophoresis

(a) Preparation of a 1% agarose gel

1. The gel casting tray was rinsed and dried with 95% ethanol.
2. The casting tray was set on a level surface.
3. The level of the comb was adjusted with a few mm of space between teeth and the
tray that allowed wells to form in the agarose,
4. 1 gram of agarose powder was weighed out and suspended in 100mL 1X TBE
buffer.
5. To dissolve the agar, the solution was boiled in a microwave.
6. The agar was allowed to be cool on the bench until ~45oC.
7. 2µL of gel star solution was added.
8. 25-30mL agarose was poured into the casting tray.
9. The gel was allowed to solidify, the comb and tape was removed.

(b) Loading and running the gel

1. 2µL of loading dye was added to each sample.
2. The casting tray was inserted into the electrophoresis chamber with the wells
closest to the negative (black) electrode.
3. 1XTBE was gradually added to the chamber until the buffer covered the top of the
gel.
4. The sample carefully loaded into the individual wells.
5. The cover was closed, the electrodes connected and the gel was ran at 90V for 60
minutes.
6. Electrophoresis run until the bromophenol blue has migrated to ¾ of the positive
electrode end of the gel.
7. The power was shut off, the leads and the power supply unplugged. The gel
casting tray was lifted from the chamber.
 (c) Staining and photographing the gel
1. The gel was removed from the casting tray and placed on the UV light.
2. The DNA bands on the gel were observed.
3. By using camera, the picture was taken.
4. The size of the DNA band was estimated by compared with the known sizes of the
DNA ladder.

RESULT
DISCUSSION
A plasmid is a special form of DNA found in bacteria. What makes it special is that it is
circular in shape. Bacteria use plasmids to share DNA with other bacteria. There are methods
that commonly used in biochemistry and molecular biology to isolate a DNA plasmid from
bacteria and one of them is by using agar gel electrophoresis. The functions of agar gel
electrophoresis is to separate DNA and RNA fragments according to length are used to
estimate the size and charge of the DNA and RNA fragments or to protein by size.

The experiment begins with the preparation of plasmid DNA extraction which is the
plasmid was taken by the previous experiment. In this experiment, the plasmid was added
with five different buffers respectively according to the procedure with the time that has been
set for each step. Then, in certain steps, the plasmid was centrifuged so that the supernatant
has been discarded and was incubated in the time that has been set for each step. After the
plasmid DNA was extracted, the plasmid DNA was transferred to the agar gel electrophoresis
by using the micropipette and screening under the UV light and the result was captured and
recorded. The result was been compared with the ladder.

The DNA plasmid was successfully extracted from the E.coli cells and then the DNA
was successfully separated according to size by using the agarose gel electrophoresis method.
Based on the theory, buffering agent that used in this experiment maintains a constant pH,
protect the DNA from DNAses which are degradative enzymes; also binds divalent cations
that are necessary for DNAse activity. It is also neutralizes the solution, allowing the DNA
strands to crenature. The E. coli chromosomal DNA is also precipitated. The plasmid DNA
remains in the solution. The viscosity of this is very high as it has a very gel like texture.
When the supernatant is placed in a new eppendorf tube after 5 minutes of centrifuge this
causes the plasmid DNA to separate from the cellular debris and chromosomal DNA in the
pellet.

The bromophenol is then added which pulls the plasmid out and causes it to precipitate
nucleic acids. After centrifuge a small white pellet was observed at the bottom of the tube
after the supernatant was carefully removed this further purifies the plasmid DNA from
contaminants. All these changes that were observed after the addition of these solutions were
expected as they are what help us extract the DNA plasmid for an end product. After placing
the DNA plasmid in the wells electrophoresis was carried out. The results were then obtained
and recorded. The size of the DNA fragment is determined from its electrophoretic mobility.
POST-LAB QUESTION
1. Define plasmid

 Plasmid is a genetic structure in a cell that can replicate independently of the

chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium
or protozoan.

2. Plasmid naturally exists in bacterial cells. List the advantages of having plasmid.

 Plasmids have a wide range of lengths, from roughly one thousand DNA base pairs to
hundreds of thousands of base pairs.
  Bacteria can also transfer plasmids to one another through a process called
conjugation.
  Plasmids used as tools to clone, transfer, and manipulate genes.
  Plasmids contain genes that help the host to digest unusual substances or to kill other
types of bacteria.
 Plasmids can copy themselves independently of the bacterial chromosome, so there
can be many copies of a plasmid even hundreds within one bacterial cell.
 Plasmids help bacteria to survive stress

3. Differentiate between plasmid and chromosomal DNA.

Plasmid Chromosomal DNA

1. A small, circular, double- stranded DNA 1. A molecule that carries the genetic
molecule that is distinct from cell’s information in all cellular forms of life.
chromosomal DNA
2. Not considered as genomic DNA as it is 2. A type of genomic DNA
form of extra chromosomal DNA
3. Naturally occurs only in prokaryotes 3. Occurs in both eukaryotic and prokaryotic
cells
4. Circular 4. Linear in prokaryotes and circular in
eukaryotes
5. Can replicate independently from the 5. Replicated along with the genome
genome
CONCLUSION
Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids
and nucleic acids. The experimental procedures carried out were a success, the DNA plasmid
was obtained and the agarose gel electrophoresis resulted with in a clear picture and outlined
above, of the DNA being successfully separated.

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