Determining DNA Concentration by Quantitative Spectrophotometric Measurement

Many compounds absorb light in the ultraviolet or visible range of the electro-magnetic spectrum. The wavelength at which the light is absorbed is a function molecular structure of the compound. DNA absorbs in the ultraviolet range, at a wavelength of about 260 nanometers (nm); it absorbs at this wavelength because of the nitrogenous bases (A, G, C and T) of DNA. Measurement of absorbance is carried out in a spectrophotometer. The spec is set to measure how much light is absorbed at the optimal wavelength. It stands to reason that there is proportionality between how much of the compound is present and how much light is absorbed. If there is twice as much of the compound, twice as much light will be absorbed. The strength of the absorption is also a function of the molecular structure, and has been determined empirically for many compounds; this is known as the extinction coefficient. The relationship is described by the Beer-Lambert Law: Beer-Lambert Law: A = cl A = absorbance value (no units) = extinction coefficient (constant for each substance, units of M-1 cm-1) c = concentration of substance (units of M) l = light path length (in cm); all specs in common use have l = 1 cm

In the case of most compounds, the units involve molarity. But consider this: suppose you have two solutions of DNA (same volumes), one contains a millimole of DNA that is 1000 basepairs long, and the other contains a millimole of DNA that is 2000 basepairs long. Wouldnt you expect the latter solution to absorb more light, since it has more nitrogenous bases? Well, it does. So in the case of polymers, where molarity does not depend on length, it is more useful to employ units of mass per volume instead of molarity (moles per volume). For DNA spectrometry, the units of concentration are typically g/ l and the extinction coefficient has units of (g/ml)-1. For DNA, the extinction coefficient is 0.020 per g double stranded DNA per ml of solution per cm of light-path, or 0.020 per g/ml-cm. This is the same as saying a reading of 1.0 at 260nm corresponds to 50 g/ml of double-stranded DNA. Determining DNA purity by determining A260/A280 Because proteins can contaminate the DNA prep, and proteins also absorb in the ultraviolet, using A260 to calculate the concentration of DNA may give deceptively high results. We can reassure ourselves that the contamination by protein is not significant by measuring the absorbance of our DNA prep at 280 nm, because this is the wavelength at which the aromatic rings on tryptophan and tyrosine absorb (phenylalanine absorbs at 260, but never mind). As a useful rule, if the absorbance of our prep at 260 is more than 1.75 times the absorbance at 280, the DNA should be pure enough to proceed. If the ratio is greater than 2.1, you are probably not working with

DNA! If the ratio is less than 1.75, the DNA is badly contaminated with protein and may not behave well in subsequent experiments. PROCEDURE 1. Turn on the spectrophotomer about 10 minutes ahead of time to let the H2-deuterium lamp (for UV) warm up. You will take readings at 260 and 280 nm. 2. The DNA is in EB (elution buffer, 10 mM Tris, pH 8.5) or some other (usually low-salt) buffer. You will dilute the DNA by some factor in order to make the measurement otherwise you would use the entire sample up! The first thing you must do is figure out a way to ensure that the buffer does not contribute to the absorbance you measure for your DNA samples. One way is to zero the spectrophotometer against pure water and then measure the absorbance of EB at the dilution factor you will be using for your DNA. (Hint: it should be zero!) 3. Dilute a portion of the DNA sample in pure water freshly obtained from the distilled water tap. Make sure that the final volume is 1.0 ml. Deliver this to a methyl acrylate cuvette (disposable) or a quartz cuvette (which is not disposable!!!). Zero the spec at 260 nm then read the absorbance of your diluted DNA samples. Zero the spec again at 280nm and read the sample at 280 nm. Record your A260 and A280 measurements and the ratio of A260/A280. 4. Calculate the DNA concentration in your diluted sample using the Beer-Lambert law. Then calculate the DNA concentration in the miniprep.