Spectrophotometric Nucleic Acid Quantitation

  1. Determine the theoretical extinction coefficient (ε) at 260 nm (OD260) of your nucleic acid sequence in units of Mľ1 cmľ1:
    • For short oligonucleotides, extinction coefficients can vary quite a bit depending on exact base sequence. It is best to use the calculator at IDT to get a more accurate value for your sequence that reflects its composition and some nearest-neighbor base effects. Concentrations of oligos are usually measured in ÁM or nM (1 ÁM = 1 Ámol/L = 1 pmol/Ál).
    • For large PCR fragments > 500 bp, plasmid DNA, or transcripts, an approximate extinction coefficient is usually used based on the length in base pairs (assuming of 25% each base) and concentration is measured in ng / Ál:
      • Double-stranded DNA: 50 cm-1 (ng / Ál)-1
      • Single-stranded DNA: 33 cm-1 (ng / Ál)-1
      • Single-stranded RNA: 40 cm-1 (ng / Ál)-1
    • 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 wavelength 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 can help you diagnose common problems, such as too much salt in your sample or protein contamination.

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. Expect these concentration calculations to be accurate to within ~20%. The theoretical extinction coefficients are only accurate to within this range. Furthermore, 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 completely denature it.

Further Information

* Nanodrop Protocol on OpenWetWare

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Topic revision: r3 - 14 Apr 2012 - 14:43:26 - Main.JeffreyBarrick
 
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