Estimation of DNA and its purity check using UV spectrophotometer (A260 measurement)

BACKGROUND

Estimation of DNA can be done by using various method like absorbance, agarose gel electrophoresis etc. Among all estimation using spectrophotometer is convenient and widely used. It requires very less equipment. The absorbance readings are taken at 260 nm then it is adjusted for turbidity by measuring the absorbance at 320 nm and multiplying it with dilution factor and using the relationship that A₂₆₀ of 1.0 = 50 ug/ml pure DNA.

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Similarly, Concentration (µg/ml) = (A₂₆₀ reading – A₃₂₀ reading) × dilution factor × 50 µg/ml. And DNA yield (µg) = DNA concentration × total sample volume (ml) But DNA is not the only molecule that absorbs UV light at 260 nm, RNA also has great absorbance at 260nm and then there are several other contaminants like aromatic amino acids. If they are present they will contribute to the total absorbance at 260 nm.

Then there is also guanidine that can result in higher 260nm absorbance. For evaluation of purity of DNA it is better to measure absorbance from 230nm to 320nm for detection of other possible contaminants. The common purity calculation is the ratio of absorbance at 260 nm divided by reading at 280 nm. DNA which are of good quality will have the ratio between 1.7-2.0. A reading less than 1.6 will have more contaminants. So, the basic objective of this experiment is to estimate DNA and perform its purity check by using a spectrophotometer.

REQUIREMENTS

         DNA sample

         TE buffer

         UV spectrophotometer                        

PROCEDURE

At first take 10 ul of DNA sample and mix it with TE buffer. Now make dilution of the sample by a factor of 100 that is by adding 10 microliters of the sample in 990 microliters of TE buffer. After doing this transfer this to spectrophotometer for measuring the OD at A260 and A280. Estimate the quantity of DNA by using the formula.¹

                   Total DNA (ug) = (A260) (50 ug/ml/A₂₆₀) (100) (0.1 ml) ²

Where, 100 is the dilution factor and 0.1 ml is the total volume of the DNA

For checking purity of DNA take OD of the sample at 260 nm and 280 nm. If the solution contains pure DNA then OD at 260 nm will be double of OD at 280 nm and if there is any contaminant then the OD ratio will decrease.³

CONCLUSION

By this experiment we can easily estimate the amount of DNA and check its purity.

REFERENCES

1. Bailiff, D. M., and D. M. Karl. 1991. Dissolved and particulate DNA dynamics during a spring bloom in the Antarctic Peninsula region, 1986–87. DeepSea Res. 38:1077–109
2. Dortch, Q. F., T. L. Roberts, J. R. Clayton, and S. I. Ahmed. 1983. RNA: DNA ratios and DNA concentrations as indicators of growth rate and biomass in planktonic organisms. Mar. Ecol. Prog. Ser. 13:61–71.
3. Dortch, Q. F., J. R. Clayton, S. S. Thoresen, and S. I. Ahmed. 1985. Nitrogen storage and the use of biochemical indices to assess nitrogen deficiency and growth rate in natural plankton populations. J. Mar. Res. 43:437–464.

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