Monday, June 11, 2012

FAQ: Why are my DNA concentration readings at absorbance 260 nm quite different from my results when using a 1-cm cuvette spectrophotometer?

The most probable answer is a different path length was used to do the calculation. Absorbance DNA quantitation measurements are dependent on Beer’s law, A=bec, where A is absorbance in O.D., b is the path length of light in cm, c is the concentration in molarity, e is the molar absorptivity or extinction coefficient (M-1 cm-1). For a standard cuvette spectrophotometer the path length is 1 cm, but for a microplate the path length changes depending on the microplate (96, 384, etc.) and volume used. In the BMG LABTECH Control software, be sure to use the path length correction feature in order to obtain measurements that are similar to a 1-cm cuvette spectrophotometer.

Other reasons could be:
  • Measurement was performed at wavelength other than 260 nm
  • Minimum volume was not used (at least 1/3 of total well volume)
  • The data was not blank subtracted
  • UV transparent plates were not used
  • Extinction coefficient other than 0.020 M-1 cm-1 (or 50 µg/mL at 1 O.D.) was used 

 For more information on the Path Length Correction feature, visit: http://www.bmglabtech.com/technology/path-length.cfm

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