DNA Quantification and Quality Analysis

After isolation of DNA, quantification and analysis of quality are necessary to ascertain the approximate quantity of DNA obtained and the suitability of DNA sample for further analysis. This is important for many applications including digestion of DNA by restriction enzymes or PCR amplification of target DNA. The most commonly used methodologies for quantifying the amount of nucleic acid in a preparation are: (i) gel electrophoresis; and (ii) spectrophotometric analysis. If the sample amount is less, the former method is usually preferred. A. Agarose Gel Electrophoresis for DNA Quantification and Quality Analysis This method of quantification is based on the ethidium bromide fluorescent staining of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the stacked bases. The fluorescent yield of the dye:DNA complex is much greater than the unbound dye. UV irradiation at 254nm is absorbed by the DNA and transmitted to the dye and the bound dye itself absorbs radiation at 302nm and 366nm. This energy is retransmitted at 590nm, the reddish-orange region of the visible spectrum. In case of plant genomic DNA, the nucleic acids are electrophoretically separated on a 0.7-0.8% agarose gel containing ethidium bromide at a final concentration of 0.5 µ g/ml. The quantity of DNA can be estimated by comparing the fluorescent yield of the samples with a series of standards, for instance, lambda (λ ) DNA at varying known concentrations. This provides a very rapid and sensitive means of estimating the nucleic acid concentration. A large number of samples with as little as 1-5ng of DNA can be quantified. Besides quantification, it also allows provides the advantage of analyzing the quality of the DNA preparation. Native DNA, which migrates as a tight band of high molecular weight (≥ 40 kb), presence of RNA, and degraded/sheared DNA, if any, can be visually identified on the gel. Procedure 1. Prepare a 0.8% agarose gel. 2. Add 1 µ l of 6X gel loading dye to 2-3 µ l of each DNA sample before loading the wells of the gel. Addition of dye allows us to note the extent to which the samples might have migrated during electrophoresis, so that it can be halted at an appropriate stage. 3. Load at least 1 or 2 wells with uncut, good quality λ DNA or any previously quantified DNA samples (50ng and 100ng) as molecular weight standards. 4. Run the submarine electrophoretic gel at 70V till the dye has migrated one-third of the distance in the gel. 5. DNA can be visualized using a UV transilluminator and quantified in comparison with the fluorescent yield of the standards. Note: For SSR and RAPD analysis, it is more important to have good quality DNA samples (unsheared/undegraded DNA), than high quantities of DNA. In contrast, RFLP analysis requires larger quantities of DNA, since the technique is not PCR-based. B. Spectrophotometric Determination Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show

absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents. The ratio of OD260/OD280 should be determined to assess the purity of the sample. This method is however limited by the quantity of DNA and the purity of the preparation. Accurate analysis of the DNA preparation may be impeded by the presence of impurities in the sample or if the amount of DNA is too little. In the estimation of total genomic DNA, for example, the presence of RNA, sheared DNA etc. could interfere with the accurate estimation of total high molecular weight genomic DNA. Procedure 1. Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as 280nm. 2. Add 10 µ l of each DNA sample to 900µ l TE (Tris-EDTA buffer) and mix well. 3. Use TE buffer as a blank in the other cuvette of the spectrophotometer. 4. Note the OD260 and OD280 values on spectrophotometer. 5. Calculate the OD260/OD280 ratio. Comments:  A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.  A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.  A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol. In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA. 6. The amount of DNA can be quantified using the formula: DNA concentration (µ g/ml) = OD260 x 100 (dilution factor) x 50 µ g/ml 1000 Spectrophotomteric Conversions for Nucleic Acids: 1 A 260 of ds DNA = 50 µ g/ml 1 A 260 of ss oligonucleotides = 33 µ g/ml 1 A 260 of ss RNA = 40 µ g/ml Reference Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols: CIMMYT Applied Biotechnology Center. Second Edition, Mexico, D.F.: CIMMYT.

Laboratory for Microbial Ecology

Department of Earth, Ecological and Environmental Sciences University of Toledo

DNA quantification (spectrophotometry)
Materials DNA standard solutions We have a standard series of herring sperm DNA solutions that includes DNA concentrations of 500, 100, 50, and 10 ng DNA μl-1. Keep stocks of these solutions by diluting the concentrated herring sperm DNA (10 mg ml-1) accordingly in DNase/RNase-free water. D.I. water for dilution of DNA prior to reading. One sterile 1.5 ml microcentrifuge tube for each sample Plastic (for concentrated samples, >50 μg ml-1) or quartz (for low concentration samples, < 50 μg ml-1) cuvettes. Spectrophotometer • Wear gloves throughout the entire protocol. • Do not cross-contaminate your samples or the solutions. Be aware of your pipette tip. • Work clean, either on fresh blue bench paper, in the hood, or on a freshly ethanol treated bench top. • Perform all centrifugations with the hinge of the tube pointing “up”. • Do not use a vortex at any point in this protocol unless specified. Begin by turning on the spec to allow the lamp to warm up for approximately 15 minutes before reading samples. Be sure that there is no cuvette in the well as the spec goes through its self-diagnosis.

The protocol in brief You will quantify DNA in solution by measuring the absorbance of light (260 nm) in a spectrophotometer. A. Preparing DNA samples 1. Remove the standards and your samples from the freezer and thaw them on ice or in the refrigerator. Mix them by tapping the side of the tube with your finger. Do not vortex to mix. 2. In a separate sterile 1.5 ml microcentrifuge tube for each standard/sample, mix 10 μl of DNA with 990 μl of D.I. water. Vortex to mix. Let this solution stand for 10 minutes to ensure the complete diffusion of DNA throughout the solution. This represents a 1:100 dilution of the standards and your DNA samples. B. Preparing the spectrophotometer
A typical spectrophotometer

3. Select “DNA/RNA”, and follow the cues given by the spec to set the machine up for your readings: We most commonly quantify: dsDNA

Absorbance of 1 relates to: 50 μg ml-1 DNA 4. Press “Enter” to validate these settings. 5. Select “Dilution factor” and enter “100” to account for the DNA concentration in your sample being 1:100 of your original stock. 6. Press “Enter” to validate these settings. 7. Blank the spec by first inspecting the cuvette to make sure that there are no smudges or blemishes that might interfere with the absorbance reading and then inserting a sample of water identical to the water in which you diluted your DNA sample and pressing “Read blank” 8. Return to the assay screen by pressing “>”. The spec is now set to begin reading standards and samples. C. Reading standards/samples 9. Inspect the cuvette to make sure that there are no smudges or blemishes that might interfere with the absorbance reading. Briefly vortex the DNA sample again and transfer the solution to the cuvette being careful not to form bubbles along the wall of the cuvette. First read the standards. 10. Insert the cuvette into the spec. Be sure that the correct face of the cuvette is aligned with the direction of the light beam. Close the lid and press ”Read sample” to begin reading. An absorbance reading will appear on the screen when the reading is complete (approximately 3 seconds). 11. Prepare the next standard as above, and continue reading until all standards have been quantified. The readout will provide several important pieces of data: “A260” – this is the wavelength of light that is absorbed by DNA. This value is used to determine that concentration of DNA in your sample according to the conversion factor you set previously (A260 of 1.0 = 50 μg ml-1 DNA, step 3). “A280” – The absorbance generated at 280 nm is used in the ratio A260:A280, which determines the purity of the DNA. Samples are considered of adequate purity if A260:A280 >1.5. “Conc.” – Based on the conversion factor and dilution factor you programmed into the spec at start-up, this is the concentration of DNA in your sample (μg ml-1). The results of the standard readings should be accurate (± 10%) before proceeding with your samples. 12. Continue reading your samples as above until all samples have been quantified. 13. To print your results, press “Print”, at which time you will be given three printing options. Choose option “3”, which directs the spectrophotometer to print all unprinted sample data. The other two options can also be used but check the manual to determine if these are the best options. 14. To exit the program press “Cancel”, then “<” to “Exit assay”. TURN THE SPEC OFF WHEN YOU ARE FINISHED.
DNA quantification, 10/2004 3

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