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BIOCHEMISTRY
RESEARCH ARTICLE

Isolation and Relative Quantification Nucleic Acid of Musa acuminata Using Absorbance
Spectrophotometry
Aplacador, Luis Bernard; Cabrera, Joseph; De Guzman, Mark Anthony; De Tomas, Xena;
Salazar, Rodney
Bachelor of Science in Chemistry 3-2, S.Y. 2014-2015, Polytechnic University of the
Philippines

Abstract (Arial black, 11)
Body (Arial Unicode MS, font 10)
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Keywords (TNR,bold; 11): example (TNR, font 10)
Introduction
Nucleic acids quantification is
important in many research procedures.
Due to factors such as the purity of the
sample, it can be difficult to measure
accurate concentration of DNA or RNA in
the sample. DNA and RNA maximum
absorption is at 260 nm and proteins
absorb strongest at 280 nm. However,
50%-55% of the absorbance at 260 nm of
nucleic acids also absorb significantly at
280 nm.
DNA molecules are long, slender
molecules that carry the heritable
information of organisms on to future
generations. Because of their size, it is
impossible to see a single DNA molecule
with the naked eye. It would take about
300,000 DNA molecules side by side to
make a bundle as thick as a human hair.
When subjected to certain conditions, it is
possible to collect “large” amounts of DNA
to make it visible.

Polytechnic University of the Philippines
BS CHEMISTRY - THIRD YEAR STUDENTS
S.Y. 2014 - 2015

In many cases, absorbance
spectrophotometry is the most rapid and
accurate approach for determination of
concentration of relatively pure nucleic
acids. The amount of light the sample
absorbed is directly proportional to the
concentration of the nucleic acid in the
sample.
Spectrophotometry is a vital,
versatile tool that is common to almost
every aspect of biological, chemical and
molecular biological research. The speed
at which concentration are known can
minimize the time required for routine
measurement of purified samples. This is
particularly important for mRNA isolation
for cDNA synthesis and for quantification
of oligonucleotide primers for PCR or
sequencing.

Materials and Methods
I.

Cell Disruption/Cell Lysis

Volume 1, Issue 1, reference number

The mixture can be divided for consumption and for experiment purposes.5 ml was diluted to 4ml for preparation for the absorbance reading. Using concentrated alcohol solution of 90%. The results of the reading was then recorded and analyzed. which increases the formation of precipitate in polar ethanol. residual organic Volume 1. The soap from the buffer solution disrupts the cell membrane and nuclear membrane’s phospholipid bilayer by reacting with the phosphate group of the phospholipid. an aliquot portion was placed into a test tube until the volume reaches ¼ of the test tube’s height. buffer solution has created it was again homogenized using fruit blender.” shielding some of the negative charge of the nucleic acid. after which they were air dried and then reconstituted into 3 mL of distilled water. Results and Discussion Mechanical disruption of Musa acumita cells were achieved using a fruit blender. Once the components are released. by hydrogen bonding it can break up the “hydrated shell” in nucleic acid phosphate. This entire process was done in triplicate. the Na+ and Cl. A wavelength scan of the three samples with 260 nm and 280 nm was carried out using the Hitachi U-5100 UVVis Spectrophotometer with distilled as the reference. A buffer solution made from detergent and salt (NaCl) is mixed with the cells.Page 2 of 8 Three bananas crushed ice and distilled water were purée in fruit blender until smooth (homogenous) was formed. Presence of ice lowers the activity of enzymes that could break it apart. making it less hydrophilic.Y. After some time white/clear precipitates were observed these aggregates were then spooled out of the ethanol layer into a watch glass using an inoculating loop. Furthermore. an ethanol precipitation process was carried out by overlaying the mixture in the test tube with 90-95% ice cold ethanol and then incubated in an ice bath for 15 Sample Preparation and Absorbance Reading After reconstitution. the process that exposed its cell membrane. Issue 1.ions in the buffer binds to several of the negatively charged phosphate groups of the nucleic acid’s sugar-phosphate “backbone. 2014 . an aliquot from the three solution of 1. reference number . Then. Ethanol precipitation enables the removal of alcohol soluble salts. allowing the formation of ionic bond between cations. II.2015 Ethanol precipitation In mixture the phosphates in nucleic acid were surrounded by the water molecule (hydrolysis) which prevents its interaction with cations. A mixture for experiment was added a few drops of Joy Lemon Dishwashing and 2-5g of NaCl. The neutralization of nucleic acid makes it more non-polar. Polytechnic University of the Philippines BS CHEMISTRY . III. DNA Purification and Reconstitution From the mixture with added detergent. this releases the cellular components into the buffer. minutes.THIRD YEAR STUDENTS S. It is important that alcohol and mixture do not mix.

2015 Volume 1. See figure 1. protein impurities in nucleic acid could be removed by addition of chaotropic salt in the solution. Test tubes were dipped in ice bath to increase the rate of formation of precipitates called nucleoprotein. See figure ().265(±0.Page 3 of 8 solvents and detergents.046(±0.003) 0. reference number .375.038(±0.003) Mean 0. Issue 1.Y. after the removal of ethanol precipitates were dissolved in distilled water then obtained dilution factor of 0.256(±0.004) 0.THIRD YEAR STUDENTS S. DF= V 1 1.5 ml = =0.4 V 2 4 ml Figure 1 Set ups were subjective to low temperature to decrease solubility of nucleic acid in ethanol Absorbance(nm) Trial 260 280 1 0.003) 0.030(±0. 2014 .052(±0.003) 3 0.035(±0.003) 2 0.044(±0.005) Polytechnic University of the Philippines BS CHEMISTRY .003) 0.

Issue 1. 2014 . DNA concentration is estimated by measuring the absorbance at 260nm. Air drying let alchohol leaves the precipiate Table 1 Absorbances at 260 and 280 nm See table ().2015 multiplying by the dilution factor. Values in trial 3 are not included in the calculations for Mean of the absorbances for both 260 and 280 nm for its large deviation from the other values. Values for the third trial are not included in calculations of Least squares Concentration (µg/ml) = (A260 reading) × dilution factor × 50µg/ml Volume 1.THIRD YEAR STUDENTS S.Page 4 of 8 Figure 2. Polytechnic University of the Philippines BS CHEMISTRY .Y.0 = 50µg/ml pure dsDNA. this may considered a random error. reference number . and using the relationship that an A260 of 1. See Graph ().

6000000000000003E-2 DNA Concentration(ug/ml) Graph 1 Concentration vs Absorbance at 260nm(trial 3 were not included in getting the least square for its large deviation from other values) A260/A280 It can be difficult to accurately measure the concentrations of DNA. reference number .8000000000000004E-2 6. RNA and protein in complex mixtures.THIRD YEAR STUDENTS S.02 0. Issue 1.02x + 0.06 0. 2014 . measuring absorbance at 260 nm and at 280 nm can provide validation of the purity of nucleic acid samples.01 0 9.07 R² = 1 0. Pure nucleic acid samples would have an A260/A280 ratio of 2.0. while protein Polytechnic University of the Philippines BS CHEMISTRY .Page 5 of 8 0.03 0.05 0.Y.2015 Volume 1.04 Absorbance at 260nm f(x) = .0. However.

2(±0.052(±0.003) 1. Absorbance(nm) A260/A280 Remarks Trial 260 280 1 0. reference number .038(±0.030(±0.1) Contaminated 2 0.256(±0.1(±0.046(±0.265(±0. Polytechnic University of the Philippines BS CHEMISTRY .044(±0.02) Contaminated Mean 0. 2014 .Y.003) 0.003) 0. Issue 1.003) 1.THIRD YEAR STUDENTS S.Page 6 of 8 would be 0. lower ratio values indicate the presence contaminants.005) Conclusion References   All tables and figures won’t follow the two-column rule References: ACS format .2) Contaminated 3 0.004) 0.04(±0.035(±0.57.003) 0.003) 1.2015 Volume 1.

2014 .2015 Volume 1. reference number .Y.THIRD YEAR STUDENTS S. Issue 1.Page 7 of 8 Polytechnic University of the Philippines BS CHEMISTRY .