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UV Spectrophotometric Analysis of DNA and RNA

The concentration of an RNA or DNA sample can be checked by the use of UV spectrophotometry. Both RNA and DNA absorb UV light very efficiently making it possible to detect and quantify either at concentrations as low as 2.5 ng/l. The nitrogenous bases in nucleotides have an absorption maximum at about 260 nm. Using a 1-cm light path, the extinction coefficient for nucleotides at this wavelength is 20. Based on this extinction coefficient, the absorbance at 260 nm in a 1-cm quartz cuvette of a 50g/ml solution of double stranded DNA or a 40g/ml solution of single stranded RNA is equal to 1. You can calculate the concentration of the DNA or RNA in your sample as follows:

DNA concentration (g/ml) = (OD 260) x (dilution factor) x (50 g DNA/ml)/(1 OD260 unit)

RNA concentration (g/ml) = (OD 260) x (dilution factor) x (40 g RNA/ml)/(1 OD260 unit)

In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm, due mostly to the tryptophan residues. The absorbance of a DNA sample at 280 nm gives an estimate of the protein contamination of the sample. The ratio of the absorbance at 260 nm/ absorbance at 280 nm is a measure of the purity of a DNA sample; it should be between 1.65 and 1.85.

Phenol has an absorbance maximum of 270 but the absorbance spectrum overlaps considerably with that of nucleic acids. If there is phenol contamination in your DNA sample, the absorbance at 260 nm will be high, giving a false measure of DNA concentration. These procedures are specific to the Beckman DU 640B spectrophotometer.

Procedure for using the to measure OD260 and OD280

  1. Turn the machine on using the switch in the back of the machine (lower right-hand side as you face the machine, turn on the monitor screen and printer. Wait for the machine to go through its start up routine.
  2. Open the lid on the top of the machine. Choose the small cuvette holder and place it in the holder in front of the light source. The word FRONT should be toward the front of the machine.
  3. Click on the UV light key to turn the UV on. It takes about a minute for the UV lamp to warm up. Quit the diagnostic screen by clicking on the word QUIT in the upper right hand corner. The Main Menu will appear Choose Nucleic Acid from the menu. When the nucleic acids analysis menu appears, make sure that the measured absorbances are 260 and 280. Do not use the calculation functions.
  4. Use lens paper to clean the surfaces of the cuvette. Rinse the cuvette chamber with 70% ETOH. Be sure to remove all of the ETOH after the wash. Place 100l sample of your blank (nH2O, TE, whatever your DNA or RNA sample is dissolved in) in the cuvette chamber. Place the cuvette in the holder and place the lid on the holder.
  5. Shut the lid of the machine.
  6. Click READ BLANK in the bottom left corner of the screen.
  7. Prepare a 1:100 dilution of the sample you want to read.
  8. After the machine has read the blank, remove the cuvette, remove the blanking solution from the chamber, rinse and dry the chamber and place your diluted sample in it.
  9. Click on READ SAMPLES in the upper left hand of the screen.
  10. After the machine has read your sample, the data will appear on the screen. You do not need to print this each time you measure a sample. A machine will collect data for you.
  11. Between samples clean the cuvette as described above. You do not need to blank each time unless your samples are dissolved in different solutions. If you use a different cuvette, you must run a blank.
  12. When you are finished reading samples, remove and clean the cuvette and put it away. Print your results by clicking on PRINT at the top right of the screen. QUIT the data screen. This will take you back to the main menu. Turn off the UV. You can turn off the machine while the Main Menu screen is active. Turn off the machine the monitor and the printer.
  13. Calculate the concentration of your RNA or DNA sample and the OD260/OD280