PROCEDURE:

PART A: DNA EXTRACTION

Part A (i): Sample preparation and Lysis Reaction
1. Every group was provided with one sample (chicken sausage). The sample
was cut and minced about 2mm2 fragment using a knife. Then, the sample
is transferred carefully into 1.5 ml of microcentrifuge tube.
2. The following are combined in the 1.5 ml microcentrifuge tube which
containing sample:
Reaction setup
10X KAPA Express Extract Buffer
1 U/L KAPA Express Extract Enzyme
PCR-grade water

Volume
(L)
10
2.0 (2 U)
Up to 100

3. After that, the mixture was vortex about 1-2 seconds. Next, the mixture
was incubated for 10 minutes at 75C. During this step, cells were lysed,
nucleases and proteins degraded and DNA was released.
4. Then, the mixture was incubated for 5 minutes at 95C to inactive the
thermostable KAPA Extract protease.
5. After that, the mixture was vortex again about 1-2 seconds before
centrifuged at maximum speed of centrifuge machine used (10000 rpm)
for 1 minute to pellet debris.
6. Then, it will form 2 layers which the supernatant (contained DNA) was
transfer to the fresh 1.5 ml microcentrifuge tube.
7. The DNA sample was ready to be analysed which 5L of DNA extract was
used for 0.8% agarose gel electrophoresis. The balance DNA extract was
stored at -20C for the next experiment.

Part A (ii): Preparation and Methods of 1% Agarose Gel Electrophoresis

1. The 1% of agarose gel was prepared and placed in the gel into the
electrophoresis tank which containing 1X TAE buffer.
2. About 5L of DNA sample with 1L 6X loading dye was mixed together by
using micropipette.
3. Then, about 3L DNA-HindIII marker was mixed.
4. The sample mixture and DNA marker was load into the wells. A voltage of
75 volts was applied for 60 minutes.
5. After the running was done, the gel was stained in the staining solution
(ethidium bromide) for 10 minutes. Then, the gel was distaining solution in
distilled water for 10 minutes.
6. The gel was observed under UV using Biorad Image Analyzer.

PART B: DNA QUANTIFICATION & QUALIFICATION

Part B (i): Spectrophotometric Determination of DNA Concentration

1. About 3L of DNA sample was diluted with distilled water and analysed at
A260 and A280.
2. The A260/ A280 ratio provides an estimate of the purity of the DNA.

RESULTS AND DISCUSSION:

This experiment involve in two parts which the first part was the DNA
extraction by using 1% of agarose gel electrophoresis and the second part was
DNA quantification and qualification by using spectrophotometric. The sample
that be used to determine the DNA is chicken sausage. The sample was lysed by
using a plastic mortar as long as the DNA in the sample is not disturb. After that,
the DNA sample was centrifuged to isolate the sample which contained DNA and
pallet. After the centrifugation method, it produce two layers of supernatant and
pallets. Thus, the DNA material was located at supernatant. The supernatant
produced was collected for the next step which was agarose gel electrophoresis
method. First of all, agarose gel electrophoresis is a process to identify and
analyse DNA and RNA strands. Besides, the agarose gel electrophoresis is an
easy way to separate DNA fragments by their sizes and visualize them. This is
done by separating the genetic material by its size. Before this method was
running, the agarose gel must be prepared. 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. Besides, in this method, the 6X loading
dye was used to coloured the DNA while the DNA-HindIII marker was used as a
positive control for this method. The genetic material which was a DNA sample is
placed in a wells of the agarose at the cathode end. From this part, Lee et. al.,
2012 reported that the negatively charged nucleic acid molecules in the DNA
sample will move through the agarose matrix with the assistance of an electric
field which called electrophoresis. This is because the genetic material in DNA
sample is negatively charged and it will move towards the anode when the
current is supplied. Mohamed, 2012 reported that the amount if voltage used is
crucial to the migration of the genetic material. When increasing the voltage
applied to the gel, the larger fragments migrate. Thus, the voltage applied was
75 volts for 60 minutes. After the run was shut down, the gel was then immersed
in ethidium bromide which is a fluorescent dye that covalently binds between the
bases of nucleic acid. So that, the UV light will passed through the gel to make
the genetic material visible. Ethidium bromide (EtBr) is the most common dye
used to make DNA or RNA bands visible for agarose gel electrophoresis. It is
fluoresces under UV light when intercalated DNA through an ethidium bromidetreated gel and visualizing it with UV light.

 DNA-HindIII
marker
(positive
control)

The fragment
produced that
contained
genetic
material.

The Figure 1.0 shows the fragment produced when the UV light passed through
the gel.
From the right, Lane 3 was the sample and Lane 4 was the duplicate of the same
sample.

The Figure 1.1 shows the fragment produced when the UV light passed through
the gel.
From the right, Lane 3 was the sample and Lane 4 was the duplicate of the same
sample. (The same result obtained but with different brightness and contrast.)

From the Figure 1.0 and Figure 1.1, it shows that the fragment produced
when the UV light passed through the gel and making the genetic material to
visible. Besides, from the figures it shows the clear band of DNA near the wells.
This is because of a little denaturation of DNA was occurred. Besides, a mixture
of large and small fragments of DNA that has been run through an agarose gel
will be separated by size. The agarose gel forms a porous lattice in the buffer
solution and the DNA must slip through the holes in the lattice in order to move
toward the positive pole. This slows the molecule down. Therefore, the larger
molecules will be slowed down more than the smaller molecules, since the
smaller molecules can fit through the holes easier. So, from the result obtained in
Figure 1.0 and 1.1, the upper fragments was the larger molecules of DNA but
there was not clearly shown while the lower fragments was the smaller
molecules of DNA which indicated by the second last of DNA-HindIII marker.
Thus, when the smear travelled from the upper to the bottom and it indicated
the size of the DNA. Both sample and duplicate sample of DNA has been
separate including of their size. Size of the DNA sample based on the marker
DNA-HindIII marker as the reference, range of the DNA is about 23130 b.p until
125 b.p (Lee et. al., 2012). The rest is the RNA. This is because of the RNA is
smaller than the DNA sizes. The upper fragments was not clearly shown may
cause from the contamination of DNA molecules.

For the second part in this experiment was for determination of DNA
concentration by using spectrophotometric. A spectrophotometer is employed to
measure the amount of light that a sample absorbs. This instrument operates by
passing a beam of light through a sample and measuring the intensity of light
reaching a detector. Absorption spectrophotometry is a widely used technique in
analytical chemistry based on the property of molecules to absorb light at
specific wavelengths. Anonymous, 2015 reported that the optical density (OD) of
a solution with a 1 cm path length, containing 50 g/ml of double-stranded DNA
or 40 g/ml of single-stranded DNA is 1.00 at a wavelength of 260nm. The
quality or purity of the sample can be determined by comparing the
measurements at 260 and at 280 nm (the wavelengths for which DNA and
protein absorb).

Sample

Wavelengt
h 1 (nm)
260

Sample 1

0.516

Duplicate

0.578

Averag
e (nm)

Wavelengt
h 2 (nm)
280

Averag
e (nm)

[DNA]
Concentra
tion
(ng/L)

DNA
Purity

0.409

5470

1.3374

0.385
0.547

0.433

Table 1.0 shows the result obtained for the quantification and qualification of
DNA

By referring to the Table 1.0, it shows that there are two types of
wavelength were used to determine the DNA concentration and purity. The
absorbance 260 nm was used to estimate the concentration of DNA in the
sample. This is because the DNA absorbs light most strongly at 260 nm. While,
the absorbance 280 nm is used as an indicator of protein contamination. This is
because of the tyrosine and tryptophan residues absorb strongly at this
wavelength (Mohmed, 2014). Besides, the absorbance generated at 280 nm is
used in the ratio A260 : A280 which estimates the purity of the DNA with respect to
contaminants that absorb UV light such as proteins or RNA. The ratio that more
than 1.5 was considered as an adequate while the ratio between 1.7 and 2.0
generally represents a high-quality DNA sample. Therefore, according the result
obtained, the DNA purity obtained was 1.3374 which the purity is very low
possibly due to the contamination with proteins or RNA. If the DNA sample is free
of contamination from RNA or protein, its concentration can be measured
accurately by the determination of the amount of UV radiation that absorbed by
the sample. So, the DNA concentration obtained in this experiment is 5470
ng/L. The concentration obtained was calculated as shown in Appendix. From
the ratio obtained, there was a RNA contamination occurred. RNA contamination
is a particular problem since its absorbance spectrum is practically
indistinguishable from that of DNA, making potential contaminations difficult to
detect. Thus any contaminating RNA will affect the final DNA concentration. But,

from this experiment it was successfully determine the DNA concentration and
the DNA purity.

CONCLUSION:

The objective of this experiment was to determine the DNA concentration

and DNA purity by using chicken sausage as sample. Both objectives had been
done by involving two parts of experiments which first was DNA extraction by
using 1% of agarose gel electrophoresis and the second part was DNA
quantification and qualification by using spectrophotometric. John E. Colligan,
2001 stated that agarose gel electrophoresis method was commonly used in
biochemistry, molecular biology, microbial genetics and clinical chemistry. The
basic protocol of this method was divided into three stages which first was gel
preparation with an agarose concentration followed by placement of DNA
samples into the sample wells and running of gel at a voltage for a time period
that will achieve optimal separation. For the third step, gel was stained and if
ethidium bromide had been incorporated into the gel and electrophoresis buffer,
it was visualized directly with UV light. In this case, 6X loading dye was used to
color the DNA while DNA-HindIII marker was used as a positive control. From this
experiment, the result obtained shows the visibility of fragment of DNA sample in
the gel when UV light passed through the gel and can be characterized by its
size which DNA fragment consist of larger molecules at upper fragment and
lower molecules at bottom fragment. Besides, from the result obtained, the
upper fragment was not clearly seen due to contamination of DNA while the
lower fragments was the smaller molecules of DNA which indicated by the
DNA-HindIII marker. For second part of this experiment, two types of absorbance
were used, 260nm and 280nm. Kim Y.H., 2012 reported that, spectrophotometric
was commonly used for quantitating the amount of DNA and RNA in
concentrated pure solution was due to its rapid, simple and nondestructive
procedures. The absorbance 260nm was used to detect DNA concentration in
sample because the optimum light absorption by the DNA was at 260nm. While
for absorbance 280nm, it was used as an indicator of protein contamination
because aromatic amino acids absorb strongly at 280nm. The DNA concentration
obtained in this experiment is 5470 ng/L. While, the DNA purity obtained was
1.3374 which the purity is very low possibly due to the contamination with
proteins or RNA.

REFERENCES:

Mohamed. (2012). Agarose Gel Electrophoresis. [Accessed 09- 11- 2015].

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from

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wide

https://www.scribd.com/doc/24147624/Agarose-Gel-Electrophoresis

web:

Lee. et. al. (2012). Agarose Gel Electrophoresis for The Separation of DNA
Fragments. [Accessed 09- 11- 2015]. Available from world wide web:
http://www.jove.com/video/3923/agarose-gel-electrophoresis-for-the-separationof-dna-fragments

Anonymous. (2015). Determination of DNA Concentration and Purify by

Ultraviolet Spectrophotometer. [Accessed 09- 11- 2015]. Available from world
wide web: http://people.rit.edu/~rhrsbi/GEPages/LabManualPDF5ed/09%20UV
%20Absorption.pdf

Mohmed. (2014). Quantification of DNA. [Accessed 09- 11- 2015]. Available

from world wide web: https://www.scribd.com/doc/24147687/Quantification-ofDNA