Quantification of DNA
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Mahathir MohmedQuantification of DNA
Uploaded by
Mahathir MohmedPlant Biotechnology
LABORATORY EXERCISE
Quantification of DNA
Introduction
A spectrophotometer is employed to measure the amount of light that a sample
absorbs. The instrument operates by passing a beam of light through a sample and
measuring the intensity of light reaching a detector. When a photon encounters an
analyte molecule, there is a chance the analyte will absorb the photon. This
absorption reduces the number of photons in the beam of light, thereby reducing
the intensity of the light beam.
Absorption spectrophotometry is a widely used technique in analytical chemistry
based on the property of molecules to absorb light at specific wavelengths. The
optical density (OD) of a solution with a 1 cm path length, containing 50 µg/ml of
double-stranded DNA or 40 µg/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).
The advantages of spectrophotometry usage are that the process of obtaining result
is rapid. The quality of DNA can be assessed to determine the level of degradation.
The whole procedure is relatively inexpensive, time saving and the result obtained is
very reliable. The machinery is also easy to operate as it is automatable. But, the
spectrophotometer is not human DNA specific. Presence of other microorganism
DNA will be detected as well.
Materials and Methods
1. Spectrophotometer was set to the desired wavelength to measure the
concentration and the purity of the kalanchoe Blossfeldina calandiva DNA
samples.
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2. 5 μl DNA was diluted to 495 μl distilled water in a microcentrifuge tube. It was
mixed by pipetting up and down several times.
3. Mixture was transferred into a glass cuvette using pipette.
4. Sides of the glass cuvette were wiped and placed in the spectrophotometer
chamber.
5. Lid was closed and readings were obtained for each wavelength (A230, A260 and
A280).
6. Concentration and the purity of DNA sample were determined.
Results
Wl 1 (nm) Wl 2 (nm) Wl 3 (nm)
Difference Ratio Result
260 280 230
1.315 1.136 1.673 0.179 0.667 0.179
DNA concentration
Concentration (µg/ml) = (A260 reading) × dilution factor × 50µg/ml
Concentration (µg/ml) = 1.315nm x 100 dilution factor x 50µg/ml
Concentration (µg/ml) = 6575 µg/ml or 6.575 µg/µl
DNA purity
absorbance at 260 nm = 1.315 = 1.158
absorbance at 280 nm 1.136
Discussion
Significance use of each wavelength
A260 : DNA absorbs light most strongly at 260nm. This value is used to estimate
concentration of DNA in the sample.
A280 : since tyrosine and tryptophan residues absorb strongly at this wavelength, the
absorbance at 280nm is used as an indicator of protein contamination. Absorbance
generated at 280nm is used in the ratio A260: A280 which estimates the purity of the
DNA. Samples are considered of adequate purity if A260: A280 is greater than 1.5.
A230 : high absorbances at this wavelength can be indicative of carry-over of either
guanidium salts (used to facilitate DNA binding to silica columns) and phenol (used in
phenol/chloroform extractions) that known to absorb strongly at 230nm.
Sample concentration
If a DNA sample is free of contamination from protein, phenol, agarose, or RNA, its
concentration can be measured accurately by determination of the amount of UV
radiation that is absorbed by the bases present in an aliquot of the sample. 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. RNA
contaminants can be removed simply by digestion with RNAse followed by a
purification step to remove the protein.
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Sample purity
The ratio of the readings at 260 nm and 280 nm: A260/A280; provides an estimation of
the DNA purity with respect to contaminants that absorb UV light, such as proteins
or RNA. Ratio of more than 1.5 was considered adequate while between 1.7 and 2.0
generally represents a high-quality DNA sample. According to result above, purity of
DNA obtained was considered very low possibly due to contamination with proteins
or aromatic substances such as phenol. The A260/A280 ratio is significantly influenced
by pH. Since water is not buffered, the pH and the calculated A260/A280 ratio can
significantly vary. Low pH readings with low A260/A280 ratio reduce the sensitivity due
to protein contamination. For accurate A260/A280 values, it is recommended to
measure the absorbance in a slightly alkaline buffer for example 10mM Tris-HCl, pH
7.5 and setting the spectrophotometer to zero.
Conclusion
From this experiment, we become aware and understood on how to measure
concentration and purity of DNA sample, and how to improve the outcome and
overall experiment procedures.
References
Beckmann, J.S. and Osborn, T.C. 1992. Plant Genomes: Methods for Genetic and
Physical Mapping. Kluwer Academic Publisher, Dordrecht, Netherlands.
Tuffaha, M.S.A. 2008. Phenotypic and Genotypic Diagnosis of Malignancies: An
Immunohistochemical and Molecular Approach. German-Jordan Center for
Laboratory Medicine, Jordan.
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(071009)
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