CEB PRACTICAL 5

LAB REPORT TITLE DNA AMPLIFICATION AND

PREPARATION OF AGAROSE
GEL AND ANALYSING DNA
FRAGMENTS
DAY OF PRACTICAL 7 MAY 2019

DUE DATE 13 MAY 2019

NAME JENNIFER A.

GROUP A

1. Title
DNA amplification and preparation of agarose gel and analyzing DNA fragments.
2. Introduction
Agarose gel electrophoresis is a process that undertakes biochemistry and molecular biology understandings
to identify and analyze 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. 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 centimeter 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.
2.1 Hypothesis
If gel electrophoresis separates and sorts fragment by size and electric charge, then the
size of the DNA sample can be estimated to confirm the PCR was successful.
2.2 Purpose of the study
To determine the presence or absence of PCR products and quantify the size (length of
the DNA molecule) of the product.
3. Methods and materials
First of all, an agarose gel was created by suspending dry agarose in a buffer solution. It was
boiled until the solution became clear. The result was a flexible gelatin-like substance.

FIGURE 1. Preparation of Agarose gel

Agarose powder was poured into a borosilicate/pyrex screw capped bottle 100ml of 1X buffer
TBE/TAE was added. The substances were swirled to mix them completely then heat in a
microwave to dissolve completely. The mixture was then left to cool to about 60°C. Ethidium
bromide (10mg/ml) solution was not added to the mixture. During the cooling process the gel
 casting system was arranged to ensure that it is in a flat position using a spirit level. It was left to
solidify. After the gel was solidified, the comb was removed, carefully to prevent ripping the
bottom of the wells. While the gel was still in its plastic tray, it was inserted horizontally into the
electrophoresis tank.

FIGURE 2. 1× TBE/TAE buffer filling FIGURE 2. Bromophenol blue solution

electrophoresis tank

Then, 1X TBE/TAE buffer was filled into the electrophoresis tank until the gel was covered
by the buffer. The PCR products (DNA) was then thawed. A micropipette was used to place
2-3μl of 0.25% bromophenol blue solution on a sterile parafilm, for each sample to be
analyzed. 5μl of the PCR product was mixed with the loading dye by repeated pipetting on
the parafilm. The prepared mixture was pipetted into the sample wells. The fact that
dispensing should be done steadily without touching the gel or overflowing the well was
taken into consideration. The apparatus was closed and connected to the power pack with
the cathode connected near the samples end and it was run at 80-100 volts for 30 minutes or
until the dye has migrated for 2 cm into the gel. The power supply was turned off and
disconnected before transferring the gel into the U.V transilluminator for observation.

Finally, the gel was observed for the presence of the DNA bands as well as where the bands
appeared. Finally, the gel was removed from the illuminator and dispose appropriately.

Name Nucleic A260/ A260/A A260 A280 Nucleic Baseline Baseline Corrected Corrected Impurity Impurity Impurity Impurity Impurity Impurity
Acid A280 230 Acid Correction Absorbance (ng/uL) % CV 1 1 A260 1 % CV 2 2 2 % CV
A260
Factor (nm)
S. 513.422 1.579 0.885 10.268 6.503 50 340 0.08 423.5 2.07 Protein 0.817 15.71 Phenol 1.031 10.31
1

S. 785.165 1.51 0.823 15.703 10.402 50 340 -0.152 638.0 2.58 Protein 1.592 11.79 Phenol 1.488 14.37
2
5

4. Results

FIGURE 1. FIGURE 2. The UV Transilluminator

5. Discussion
Electrophoresis is a process that can separate amino acids, proteins and nucleic acids according
to the magnitude of the molecular charge, size of the molecules, applied voltage and shape of the
molecules (Smith 1991). The direction of the movement of the molecules depend on the net
charge of the molecules running in the support medium (Shimura & Kasai 1987). There are
many types of electrophoresis processes varying in line with the molecular type that is to be
separated such as: gel electrophoresis, Dielectrophoresis, capillary electrophoresis, paper
electrophoresis, pulse field electrophoresis and etc. Gel electrophoresis is a common easy
method which uses a small voltage of electric current to pushes the molecules though an agarose
gel filter passing them through an electric field with the aid of a buffer (Shimura & Kasai
1987). The buffer used is a salt water solution that allows the electric charges to flow through the
gel and prevents the gel from drying out during the experimental procedures (Smith 1991).
Loading dyes used in gel electrophoresis serve three major purposes. First they add density to the
sample, allowing it to sink into the gel. Second, the dyes provide color and simplify the loading
process. Finally, the dyes move at standard rates through the gel, allowing for the estimation of
the distance that DNA fragments have migrated.
The exact sizes of separated DNA fragments can be determined by plotting the log of the
molecular weight for the different bands of a DNA standard against the distance traveled by each
band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can
be compared against the unknown DNA samples. It is important to note that different forms of
DNA move through the gel at different rates. Supercoiled plasmid DNA, because of its compact
conformation, moves through the gel fastest, followed by a linear DNA fragment of the same
size, with the open circular form traveling the slowest.
In conclusion, since the adoption of agarose gels in the 1970s for the separation of DNA, it has
proven to be one of the most useful and versatile techniques in biological sciences research.
Questions and answers
1. What are some of the factors affecting migration of nucleic acid in gel?
Size of DNA molecule, agarose concentration, DNA conformation, voltage applied, presence of
ethidium bromide, type of agarose and electrophoresis buffer.

2. State the danger of preparing polyacrylamide gels.

It is more difficult to prepare than agarose gels because oxygen can inhibit the polymerization
process and acrylamide is a potent neurotoxin and must be handled with care. Polyacrylamide is
considered to be non-toxic but the gels should be handled with gloves due to the possible
presence of free acrylamide.
3. List two of the most frequently employed stains used in electrophoresis.
Ethidium bromide and Coomassie Brilliant Blue
4. State the potential hazard of using ethidium bromide
Due to the fact that ethidium bromide can bind with DNA, it is highly toxic as a mutagen. It may
potentially cause carcinogenic or teratogenic effects, although no scientific evidence showing
either health effect has been found.
5. State the net charge of DNA.
DNA is charged negative.
6. List the 5(five) categories of potential sources of error and give one example of each.

Sample contamination:
Take steps to prevent contamination by wearing gloves, using proper pipetting technique to
prevent contamination of the pipette and changing tips after each use.
Buffer:
Buffer was not diluted correctly or used the wrong concentration.
Gel:
Cool completely before adding samples.
Load appropriate amount of sample:
Too much of the target will cause a smear.
Electrophoresis:
Too low a voltage will take too long to run.

7. What are the detrimental effects of running the gel for a longer period on the
electrophoretic tank?
Longer electrophoretic runs will increase the separation between fragments. Adequate separation
is important for analysis of DNA fragments, especially those that are close in size. However, if
the electrophoresis is conducted for too long, DNA bands may migrate off the end of the gel.

8. Discuss the analytical applications of gel electrophoresis?

Electrophoresis is the use of an electrical current to manipulate protein molecules for a range of
biomedical research, diagnostic and manufacturing purposes.
DNA Analysis:
One leading use of electrophoresis is in the identification and study of DNA and DNA
fragments.
Protein and Antibody Interactions:
Another common form of electrophoresis is immunoelectrophoresis, which analyzes the
presence and behaviors of certain proteins.
Testing Antibiotics:
Electrophoresis plays a number of roles in the testing of antibiotics. One of the most common is
testing the purity of an antibiotic.
Testing Vaccines:
As with antibiotics, electrophoresis is useful in both the creation and production of vaccines.
9. What is the purpose of the DNA ladder/Marker?
They are a set of standards that are used to identify the approximate size of a molecule run on a
gel during electrophoresis, using the principle that molecular weight is inversely
proportional to migration rate through a gel
10. What were the band sizes of the various DNA samples?
The bands nor band sizes were not as visible or not visible at all because ethidium bromide was
not added to the agarose gel. Ethidium bromide is used because upon binding of the molecule to
the DNA and illumination with a UV light source, the DNA banding pattern can be visualized.
References:

Shimura, K. & Kasai, K.-I. 1987. Affinophoresis in two-dimensional agarose gel electrophoresis: Specific
separation of biomolecules by a moving affinity ligand. Analytical Biochemistry, vol. 161, pp.
200-206.

Smith, C. L. 1991. Separation and analysis of DNA by electrophoresis. Current Opinion in

Biotechnology, vol. 2, 86-91