Plant 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. 1

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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) 260 1.315 Wl 2 (nm) 280 1.136 Wl 3 (nm) 230 1.673 Difference 0.179 Ratio 0.667 Result 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.


Plant Biotechnology

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. (071009) (071009) (111009) (131009)


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