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Plant Biotechnology

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LABORATORY EXERCISE
Quantification of DNA

Introduction
A spectrophotometer is employed to measure the amount of light that a sampleabsorbs. The
instrument operates by passing a beam of light through a sample andmeasuring the intensity
of light reaching a detector. When a photon encounters ananalyte molecule, there is a chance
the analyte will absorb the photon. Thisabsorption reduces the number of photons in the beam
of light, thereby reducingthe intensity of the light beam.
Absorption spectrophotometry is a widely used technique in analytical chemistrybased on the
property of molecules to absorb light at specific wavelengths. Theoptical density (OD) of a
solution with a 1 cm path length, containing 50 g/ml ofdouble-stranded DNA or 40 g/ml of
single-stranded DNA is 1.00 at a wavelength of260nm. The quality or purity of the sample
can be determined by comparing themeasurements at 260 and at 280 nm (the wavelengths for
which DNA and proteinabsorb).
The advantages of spectrophotometry usage are that the process of obtaining resultis 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 isvery reliable. The
machinery is also easy to operate as it is automatable. But, thespectrophotometer is not
human DNA specific. Presence of other microorganismDNA will be detected as well.

Materials and Methods
1. Spectrophotometer was set to the desired wavelength to measure theconcentration and the
purity of the kalanchoe Blossfeldina calandiva DNAsamples.

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2.5 l DNA was diluted to 495 l 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 (g/ml) = (A260 reading) dilution factor 50g/mlConcentration (g/ml) =
1.315nm x 100 dilution factor x 50g/mlConcentration (g/ml) = 6575 g/ml or 6.575 g/l

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. Absorbancegenerated
at 280nm is used in the ratio A260: A280 which estimates the purity of theDNA. 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,
itsconcentration can be measured accurately by determination of the amount of UVradiation
that is absorbed by the bases present in an aliquot of the sample. RNAcontamination is a
particular problem since its absorbance spectrum is practicallyindistinguishable from that of
DNA, making potential contaminations difficult todetect. Thus any contaminating RNA will
affect the final DNA concentration. RNAcontaminants can be removed simply by digestion
with RNAse followed by apurification step to remove the protein.

Plant Biotechnology
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Sample purity
The ratio of the readings at 260 nm and 280 nm: A260/A280; provides an estimation ofthe
DNA purity with respect to contaminants that absorb UV light, such as proteinsor RNA.
Ratio of more than 1.5 was considered adequate while between 1.7 and 2.0generally
represents a high-quality DNA sample. According to result above, purity ofDNA obtained
was considered very low possibly due to contamination with proteinsor aromatic substances
such as phenol. The A260/A280 ratio is significantly influencedby pH. Since water is not
buffered, the pH and the calculated A260/A280 ratio cansignificantly vary. Low pH readings
with low A260/A280 ratio reduce the sensitivity dueto protein contamination. For accurate
A260/A280 values, it is recommended tomeasure the absorbance in a slightly alkaline buffer
for example 10mM Tris-HCl, pH7.5 and setting the spectrophotometer to zero.

Conclusion
From this experiment, we become aware and understood on how to measureconcentration
and purity of DNA sample, and how to improve the outcome andoverall 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.
http://www.newton.dep.anl.gov/askasci/mole00/mole00371.htm (071009)
http://elaney.org/wp/ari_krakowski/files/2009/08/b75labman_lab10_sm.pdf
(071009)
http://www.pubquizhelp.com/other/dnacalculator.html (111009)
http://www.cvg.ca/images/Performance_UV_Vis.pdf(121009)
http://biowww.net/detail-1361.html (131009)