Detection of contamination in DNA and protein samples by photometric measurements
contributed by Eppendorf |
Nucleic acid purification is a major application in a molecular laboratory. Purity and homogeneity of the sample are important considerations for subsequent applications. In practice, nucleic acids are purified with the help of commercially available kits which allow the separation of most other cellular components. However, the presence of proteins or other organic components in the eluate cannot be entirely ruled out. This is also directly related to the quality of the kit used. In the case where no kit is used for purification, additional contamination risks are posed by the chemicals used, e.g. phenol or ethanol from a phenol/chloroform extraction. Furthermore, this purification method typically yields all nucleic acids in a cell, not only the nucleic acid of interest. For example, a classic plasmid preparation without the RNase digestion step will yield approximately 90 % RNA . In order to ensure minimum contamination of the sample it makes sense to verify its purity by spectrophotometric measurements, fluorimetry or agarose gel. After considering the effort involved, the first method is certainly the simplest to perform.
For photometric determination of the concentration of a nucleic acid solution, absorbance is measured at 260 nm. Using a specific conversion factor, the concentration of the nucleic acid solution is calculated from the absorbance value. The following conversion factors (CF) are valid for an absorbance of 1 and for an optical path length of 1 cm:
dsDNA ≙ 50 μg/mL
RNA ≙ 40 μg/mL
ssDNA ≙ 33 μg/mL
The concentration (C) of the respective nucleic acid is determined via the following formula: C = CF x A (A=Absorbance), which results from a conversion of Lambert-Beer’s law:
A=ε*C*d <> C=1/ ε *A. The optical path length of 1 cm may be directly integrated into the coefficient of absorbance. The conversion factor CF is derived from the reciprocal value of the coefficient of absorbance (1/ ε = CF).
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