INTRODUCTION

Quantification and qualification of DNA is an essential process in handling and

analyzing DNA. This process enables people to check the concentration and purity
of DNA or RNA that presents in a sample or a solution. It is commonly performed in
molecular biology and is very useful for downstream applications like PCR or
restriction digests. There are two main approaches used by scientists to quantitate,
or establish the concentration, of nucleic acids such as DNA or RNA in a solution
which are spectrophotometric analysis and Fluorescent dye tagging. However, in
this experiment, the chosen quantification method is spectrophotometric analysis. A
spectrophotometer uses the absorbance or transmission of light through a liquid to
determine the concentration of a particular substance in that liquid. By measuring
the absorbance of a liquid, the concentration of a substance in that liquid can be
accurately estimated. In order to do this, the wavelength of light that maximally
absorbs by any particular substance must be known. In the case of nucleic acid
(DNA and RNA), the maximal absorbance is at 260nm. Protein maximally absorbs at
280nm and the ratio of nucleic acid to protein (260/280) is generally used as an
indicator of the purity of DNA samples (DNA Quantification, n.d.). The instrument
operates by passing a beam of light through a sample and measuring the intensity
of light reaching a detector. A spectrophotometer consists of two instruments which
are spectrometer that produces light of any selected color (wavelength) and a
photometer that measures the intensity of light.

Agarose gel electrophoresis is widely used in teaching and demonstrating the

key concepts in DNA science, as much as it is for research purposes. It is the most
effective way of separating DNA fragments of varying sizes ranging from 100 bp to
25 kb (Lee et.al, 2012). Typically, a DNA molecule is digested with restriction
enzymes, and the agarose gel electrophoresis is used as a diagnostic tool to
visualize the fragments. An electric current is used to move the DNA molecules
across an agarose gel, which is a polysaccharide matrix that functions as a sort of
sieve. The matrix helps "catch" the molecules as they are transported by the
electric current (Khalsa, 2010).

OBEJECTIVES
 1. To estimate the concentration of onion DNA from test tube A (blended) and
test tube B (non-blended) by U.V. absorption spectrophotometry.
2. To separate onion DNA fragments from test tube A (blended) using agarose
gel electrophoresis.

APPARATUS

Spectrophotometer, quartz cuvettes, agarose gel electrophoresis apparatus, power

supply, micropipettes and tips.

MATERIALS

Onion DNA, safe dye, loading dye, sterile distilled water.

PROCEDURES

A. Spectrophotometry:
1. The onion DNA was ready to be quantified by spectrophotometer. The OD 260
of a 1 in 100 dilution was measured. (10µl DNA + 990µl dH 2O)
2. The measurement was based on the fact that the DNA absorbency at 260nm
was 1.0 when its concentration was 50µg/ml. Thus, 50µg/1.0= Xµg/ A 260
Calculation: Concentration of DNA = (50 X A 260) X dilution factor
= (50 X A260) X 100

B. Agarose gel electrophoresis

1. Two microlitre of loading dye were transferred to a microtube containing ten
microlitre of onion DNA by using sterile disposable pipette tips for each
transfer.
2. The DNA sample and the loading dye were thoroughly mixed in each tube
before the samples were placed in the gel wells for electrophoresis.
3. The solidified agarose gel was placed into the casting tray. The casting tray
was then placed onto the central platform in the gel chamber box. The wells
 should be at the negative cathode end of the box where the black electrical
lead is connected.
4. About 275 millilitre of TE buffer were poured into the electrophoresis chamber
until it just covers the wells of the gel by one to two millilitre.
5. The lid was carefully placed on the electrophoresis chamber. The electrical
leads were then connected into the power supply.
6. The power and electrophoresis were turned on at one hundred volts for thirty
minutes. Bubbles were immediately appeared in the electrophoresis buffer.
The loading dye can be seen moving through the gel towards the positive
side of the gel chamber shortly after the current was applied.
7. After the electrophoresis was completed, the power supply was turned off and
the leads were disconnected from inputs.
8. The top of the gel chamber was removed and the gel along with the casting
tray was carefully taken off the gel chamber.

RESULTS

Table 1: The average value of absorbance of 260nm and 280nm from tube A.

Group 1 2 3 4 5 Average
Absorban 0.027 0.025 - 0.115 0.032 0.0498
ce
(260nm)
Absorban 0.024 0.017 - 0.055 Negative 0.0320
ce
(280nm)

Concentration of DNA = (50 X Average A260) X dilution factor

= (50 X 0.0498) X 100

= 249 µg/ml

Concentration of DNA = (50 X Average A280) X dilution factor

= (50 X 0.0320) X 100

 = 160 µg/ml

A 260
Purity =
A 280

0.0498
=
0.0320

= 1.56

Table 2: The average value of absorbance of 260nm and 280nm from tube B.

Group 1 2 3 4 5 Average
Absorban 0.037 0.014 0.013 0.030 0.011 0.0210
ce
(260nm)
Absorban 0.034 0.003 0.011 0.030 Negative 0.0195
ce
(280nm)

Concentration of DNA = (50 X Average A260) X dilution factor

= (50 X 0.0210) X 100

= 105 µg/ml

Concentration of DNA = (50 X Average A280) X dilution factor

= (50 X 0.0195) X 100

= 97.5 µg/ml

A 260
Purity =
A 280

0.0210
=
0.0195
 = 1.08

Ladder
Ladder

Figure 1 The visual of DNA fragments that has been separated by agarose gel
electrophoresis.

First lane: Miss Florisa’s sample

Second lane: Group 1

Third lane: Group 2

Fourth lane: Group 4

Fifth lane: Group 5

DISCUSSION

The spectrophotometer is an instrument employed to measure the amount of

light that a sample absorbs. It also operates on the Beer-Lambert Law. This law
stated the linear relationship between absorbance and concentration of an
absorbing species (Tissue, 1996). Thus, when monochromatic light (light of a
specific wavelength) passes through a solution there is usually a quantitative
relationship (Beer's law) between the solute concentration and the intensity of the
transmitted light. To simplify it, the more concentrated the specimen is, the less
light is transmitted through it (Asami, 2015). Based on the first part of the
experiment, the onion DNA was needed to be quantified by spectrophotometer. The
samples of tube A and tube B from every group were taken in order to find the
average value of absorbance for 260nm and 280nm. 10µl sample of DNA from tube
A and tube B were taken and added to 990µl distilled water or TE (Tris-EDTA) buffer.
This dilution step is taken to ensure the accuracy of the result. One milliliter of TE
buffer was taken then put in a cuvette and the spectrophotometer was calibrated at
260nm as well as 280nm. The TE buffer is used to zero the instrument or 'set
reference'.

Then, the OD260 and OD280 values on spectrophotometer for every groups
were noted. The average value of absorbance of 260nm and 280nm was calculated
and the value was substituted to the formula that was shown in the result to identify
the concentration of the DNA for both wavelengths. Based on table 1, A 260 and A280
were calculated for tube A which were 0.0498 and 0.0320 respectively. After
calculations have been made, the concentrations of DNA observed were 249 µg/ml
at A260 and 160 µg/ml at A 280. Unfortunately, for tube A, one of the groups
accidentally made a mistake that led to their solution being unsuitable to be used
and have to be excluded and the samples were only taken from the other four
groups. As for tube B, all samples were taken from five groups and the A 260 and A280
calculated were 0.0210 and 0.0195 respectively. Also, the concentrations of DNA
calculated was 105 µg/ml at 260nm and 97.5 µg/ml at 280nm. By comparing both
DNA concentrations at both wavelengths for both tubes, it can be observed that the
DNA concentration for tube B is much lower than tube A. This is due to the cell
membrane that was not disrupted in the previous experiment where the blending
step was skipped for tube B.

After that, the purity of the DNA must be calculated and the most common
purity calculation is the ratio of the absorbance at 260nm divided by the reading at
280nm. According to the results obtained, the purity of DNA based on the ratio of
average value of absorbance for 260nm and 280nm for tube A was 1.56 whilst tube
B was 1.08. Nucleic acids, depending on base composition, absorb maximally at
about 260nm due to the aromatic base moieties within their structure. For instance,
purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both
have peak absorbance at 260nm, thus making it the standard for quantitating
nucleic acid samples. The 280nm absorbance is measured where proteins and
phenolic compounds have a maximal absorbance. Similarly, the aromaticity of
phenol groups of organic compounds absorbs strongly near 280nm (Asami, 2015). A
ratio between 260nm:280nm of 1.8-2.0 denotes that the absorption in the UV range
is due to nucleic acids while the ratio that is lower than 1.8 indicates the presence
of proteins and/or other UV absorbers. The ratio of 1.9-2.0 also indicates pure RNA
whilst a ratio that is higher than 2.0 indicates that the samples may be
contaminated with chloroform or phenol (Gaikwad, n.d.). For tube A, the purity is
1.56 which indicates that the solution might contain too much proteins instead of a
good quality DNA. There might be contaminations occur during the processes or the
DNA probably has been degraded. As for tube B, there was also no pure DNA.
Instead, there are a lot of contaminants or proteins inside which causes the ratio to
be as low as 1.08. Over-quantity of proteins can affect the readings of the
spectrophotometer which can be one of the error.

For the second part of the experiment, the students were required to practice
the method for separating and visualizing DNA fragments which is the agarose gel
electrophoresis method. This technique has lots of applications. Generally, this
method allows the analyzation of DNA fragments that result from an enzyme
digestion of a larger piece of DNA to visualize the fragments and determine the
sizes of the fragments. In addition to its usefulness in research techniques, agarose
gel electrophoresis is a common forensic technique and is used in DNA
fingerprinting (Khalsa, 2010). Agarose's high gel strength also allows the handling of
low percentage gels for the separation of large DNA fragments. Molecular sieving is
determined by the size of pores generated by the bundles of agarose in the gel
matrix. In general, the higher the concentration of agarose, the smaller the pore
size. Traditional agarose gels are most effective at the separation of DNA fragments
between 100 bp and 25 kb (Lee et.al, 2012). Thus, it was proven as a very efficient
way of separating nucleic acids.
 In this experiment, ethidium bromide (EtBr) was replaced with safe dye.
Although it is commonly used as a non-radioactive marker for identifying and
visualizing nucleic acid bands in electrophoresis, it is highly toxic as a mutagen as it
can bind with DNA. It may potentially cause carcinogenic or teratogenic effects.
Besides, an acute exposure to ethidium bromide may cause irritation of the mouth,
upper respiratory tract, skin, and eyes (Ethidium Bromide, n.d.). Thus, the
alternative by using safe dye is brilliantly essential as it does not require special
handling technique or specific disposal method. To prepare the agarose gel, 0.24g of
agarose powder was weighed and 30ml of TE buffer was added. It was then
microwaved for 45 seconds and then poured into a gel tray. When it cools, the
agarose gel electrophoresis apparatus was set up and the samples were loaded as
stated in the procedures. Loading dye that was also 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 (Lee et.al, 2012).

To begin the process, a sample of the isolated DNA is loaded into a well of the
agarose gel and then exposed to an electric current. The electrical leads were then

connected to a power supply and was turned on at 100V for 30 minutes and the
fragments in the samples begin to travel from the wells to the other side of the gel.
The phosphate backbone of the DNA (and RNA) molecule is negatively charged,
therefore when placed in an electric field, DNA fragments will migrate to the
positively charged anode. Different forms of DNA move through the gel at different
rates. Since small DNA fragments migrate faster, the DNA is separated by size. The
percentage of agarose in the gel will determine what size range of DNA will be
resolved with the greatest clarity. Any RNA, nucleotides and protein in the sample
migrate at different rates compared to the DNA so the band(s) containing the DNA
will be distinct (Absorbance Methods, n.d.). In order to simply understand it, a larger
size of DNA will cause it to move slower and smaller sized DNA will travel further.
Tube A was used in this electrophoresis because it contains DNA. Based on figure 1,
it can be seen that the results obtained for every group were smeared except for
group 3. I was in Group 2 and my result was smeared. This smearing is usually the
result of poorly prepared gels, loading undiluted samples into the wells or poor
quality samples (Mayer, 2018). As the ladder is the theoretical value for the base
pair, the students could not compare their results with the ladder because they
were smeared. A ladder is a sample of DNA where the sizes of the bands are known
(Khalsa, 2010).

In order to minimize the errors that may occur in this experiment, several
safety regulations must be taken and the most important one is the use of gloves
that is very essential as it can lead to contamination of the sample if not being
used. For instance, the heat from the hand may cause the degradation of DNA when
it came in contact with the tube. Also, the students should be attentive when mixing
any solution with specific measurements. The observer must make sure that their
eyes are perpendicular whenever reading the measurement on any apparatus to
avoid systematic error. Lastly, for safety purposes, lab coat, gloves and masks
should always be worn during this practical so that no contamination can occur.
Besides, all of the apparatus used should be cleaned first by using distilled water so
that there will be no impurities left that will affect the solutions.

CONCLUSION

In a nutshell, this experiment allows the students to learn on how to estimate

the concentration of onion DNA by UV absorption spectrophotometry. The
concentrations and purity of DNA for tube A and B in each groups were successfully
calculated and observed according to the theoretical values as mentioned in the
results and discussion. Besides, the students also learned the method of separating
the onion DNA fragments by using agarose gel electrophoresis. Only tube A was
used in this separation method because there was no DNA in tube B as it was not
disrupted in the last practical. There were some errors in the results that may be
affected by several mistakes that occur when conducting the experiment.
Nonetheless, the students managed to perform the practical and the techniques
could always be improved from time to time in the future especially when venturing
the field of biotechnology.
REFERENCES

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https://www.addgene.org/protocols/dna-quantification/

Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel
electrophoresis for the separation of DNA fragments. Journal of visualized
experiments: JoVE, (62), 3923. doi:10.3791/3923

Khalsa, G. (2 April 2010). Agarose Gel Electrophoresis. ASU - Ask A Biologist.

Retrieved on 28 October 2019 from https://askabiologist.asu.edu/agarose-gel-
electrophoresis

Tissue, B. M. (22 February 1996). Beer-Lambert Law. Science Hypermedia. Retrieved

on 28 October 2019 from http://elchem.kaist.ac.kr/vt/chem-ed/spec/beerslaw.htm

Asami, P. (2015). Spectrophotometry (Quantification of Nucleic Acids). Introduction

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http://hpc.ilri.cgiar.org/beca/training/IMBB_2015/lectures/Spectrophotometry_IMBB_2
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Gaikwad, A.B. (n.d.). DNA quality: electrophoresis, spectrophotometry and

fluorometry. Retrieved on 28 October 2019 from
http://www.nbpgr.ernet.in/Portals/6/DMX/GENOMIC_ RESOURCES/DNA%20quality-
spectrophotometry%20and%20electrophoresis.pdf

Ethidium Bromide. (n.d.). Retrieved on 28 October 2019 from

https://ehs.research.uiowa.edu/ethidium-bromide

Absorbance Methods. (n.d.). Retrieved on 28 October 2019 from

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Mayer, M. (10 May 2018). What Causes Smearing in Electrophoresis?. Retrieved on

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