Spectrophotometric Nucleic Acid Quantitation

  1. Determine the theoretical extinction coefficient at 260 nm (OD260) of your nucleic acid sequence in units of M1 cm1:
    • For short oligonucleotides (single-stranded), extinction coefficients can vary quite a bit depending on exact base sequence. It is best to use the calculator at IDT to get an accurate value for your sequence that reflects its composition and some nearest-neighbor effects.
    • For longer oligos or dsDBA, an approximate extinction coefficient is usually used based on the mength in base pairs (assuming of 25% each base).
    • For oligos containing fluorescent probes or quenchers, you can alternatively use the absorption of these at their peak wavelengths. This can be particularly useful if they are well separated from the nucleic acid base peak.
  2. Using the Nanodrop or a UV/vis spectrophotometer. Record a waveleneght scan from about 200 nm to 600 nm. Do not just look at the A260 number unless you have a lot of experience with a protocol. Looking at the wavelength scan will help you diagnose common problems...

Notes

  1. A low A260/A230 ratio is generally a sign of a sample that contains appreciable salt that may interfere with further reactions or assays.
  2. These calculations generally assume that the nucleic acid molecule is completely unstructured (extended). Base stacking leads to a decrease in OD260 (hypochroism). Although rarely done in practice, to make highly accurate measurements, one would want to resuspend the sample in 12 M urea or otherwise denature it.
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Topic revision: r1 - 26 Mar 2012 - 18:47:22 - Main.JeffreyBarrick
 
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